173

Mutation Research, 236 (1990) 173-201 DNA Repair Elsevier MUTDNA 06004

The enzymology of apurinic/apyrimidinic endonucleases Paul W. Doetsch

a

and Richard P. C u n n i n g h a m b

a Department of Biochemistry, Emory University School of Medicine, Atlanta, GA 30322 (U.S.A.) and b Department of Biological Sciences, S U N Y at Albany, Albany, N Y 12222 (U.S.A.) (Accepted 12 March 1990)

Keywords: Apurinic/apyrimidinic endonucleases; AP endonucleases; AP lyases; DNA repair, enzymologyof

Summary Studies on the enzymology of apurinic/apyrimidinic (AP) endonucleases from procaryotic and eucaryotic organisms are reviewed. Emphasis will be placed on the enzymes from Escherichia coli from which a considerable portion of our knowledge has been derived. Recent studies on similar enzymes from eucaryotes will be discussed as well. In addition, we will discuss the chemical and physical properties of AP sites and review studies on peptides and acridine derivatives which incise D N A at AP sites.

AP endonucleases play an important role in the repair of D N A damage. AP sites arise spontaneously at a substantial rate (Lindahl and Nyberg, 1972; Lindahl and Karlstr~Sm, 1973) and they are also the product of D N A N-glycosylases which recognize and catalyze the removal of damaged or incorrect bases from D N A (Sakumi and Sekiguchi, this issue, p. 161). The action of D N A N-glycosylases followed by the action of AP endonucleases constitutes the incision step in a repair pathway which has been termed base-excision repair (Friedberg et al., 1978). AP endonucleases can cleave 5' to an AP site leaving a 3'-hydroxyl group, they can cleave 3' to an AP site leaving a 3'-a, fl-unsaturated aldehyde or, in at least one instance, they can cleave 3' to an AP site to yield an abasic sugar with a 3'-phosphoryl group (Spiering and Deutsch, 1986). The possible sites of phos-

Correspondence: Dr. Richard P. Cunningham, Department of Biological Sciences, SUNY at Albany, Albany, NY 12222 (U.S.A.).

phodiester bond cleavage adjacent to AP sites are depicted in Fig. 1. The first two incision events mentioned above have been shown to yield incised D N A which is not a good substrate for E. coli D N A polymerase I (Warner et al., 1980; Mosbaugh and Linn, 1982; Katcher and Wallace, 1983; Bailly and Verly, 1984). Incisions 5' to AP sites leave deoxyribose 5-phosphate as the 5'terminus of the nick. D N A polymerase I from E. coil cannot remove this group from D N A via its 5 ' - 3 ' exonuclease activity (Franklin and Lindahl, 1988) except at very high p H and protein concentration (Verly et al., 1974) and any synthesis which does occur at physiological p H results in a strand displacement reaction (Mosbaugh and Linn, 1982). The actual events which occur in vivo have not been determined, however the discovery of DNA deoxyribophosphodiesterase (dRpase) (Franklin and Lindahl, 1988) suggests that a further cleavage event may take place in the repair pathway. Deoxyribophosphodiesterase cleaves 3' to the 5'-terminal deoxyribose 5'-phosphate to generate a one nucleotide gap in D N A . This gap is

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174 5'

±NH2-

-~

U-p~ =O

HO

N O ! O-p I

=0

0

._~' u-p =o

j_ "

?I ~~: ~ H2 cO

I

/

Nil2

H~CO O:

/

N

"'~

J

C~ H pl "O-.I =O H:,C O

(A)

H2C/x / N ~ ~r~V/xHo o

/(MAaj)or Cellular

---~.- O.

A "°'PII=o

/

AP-Endonucleases

B--~o

f

3'

H2C .O~ OH

~

H

5'

C__.=~ (~ H

"o-p =0

-o_!=o O--,-o,

,

HIgH

(o)

(/~N ~ A P

I o \ / N\ ~ H=~,,

/

O ~

NH2

H=~.O~/N-- ~'N~f

Lyases

~,~-Elim. " " • Catalyst

O H 3'

s]

H~HH.o "O-PI =O I O I

N2~.,/OH "o I

C ;"

?

H,c,/O... / N---~ o

O

H

3' Fig. 1. Potential sites of phosphodiester bond cleavage adjacent to AP sites. 4 possible sites of cleavage exist, but only 2 are used by the vast majority of enzymes. AP endonucleases cleave hydrolytically at site A to yield a 5'-terminal residue of deoxyribose 5-phosphate and a 3'-terminal residue of deoxyadenosine. AP lyases cleave by a B-elimination mechanism at site C to yield a 3'-terminus which is an a,~-unsaturated aldehyde (the 3'-ester of deoxyadenosine 3'-phosphate with the 5'-hydroxyl group of (4R)-4-hydroxy-trans-2-pentenal) and a 5'-terminal residue of deoxythymidine 5'-phosphate. The bases flanking the AP site were selected arbitrarily. The base ring hydrogens have been omitted for simplicity.

efficiently repaired by DNA polymerase I and would apparently be required for efficient repair via the base-excision repair pathway. Bailly and Verly (1989a) have shown that the 5'-terrninal

deoxyribose 5'-phosphate readily undergoes an uncatalyzed fl-elimination reaction to yield a one nucleotide gap as well. The half-life of 2 h for this nicked AP site may not be sufficiently short to

175 allow for in vivo repair reactions to occur in bacteria. AP endonucleases which cleave 3' to AP sites yield an unsaturated aldehyde as the 3'terminus. Again, E. coli D N A polymerase I cannot efficiently repair this nick. The 3 ' - 5 ' exonuclease activity of polymerase I can only remove this lesion very slowly. Processing of this lesion requires that a 3'-repair diesterase cleaves 5' to the base free unsaturated aldehyde to generate a one nucleotide gap in the D N A . Some enzymes m a y be able to cleave both 3' and 5' to the AP site to yield a one nucleotide gap bordered by a 3'-phosphoryl and a 5'-phosphoryl group (Bailly et al., 1989a). A D N A phosphatase would be required to remove the 3'-phosphoryl group to provide a substrate for D N A polymerase. Thus it appears that base-excision repair requires 3 steps that result in a one nucleotide gap in DNA. In E. coli, where the most information is available, it appears that the repair of AP sites is mediated by the action of exonuclease III or endonuclease IV (AP endonucleases which cleave 5' to AP sites) followed b y the action of dRpase. While endonucleases which cleave 3' to AP sites are not a major portion of the total AP endonuclease activity of E. coli (Cunningham and Weiss, 1985), they are found associated with D N A N-glycosylase activities. Initiation of repair of some damaged bases could proceed via the action of a D N A N-glycosylase and its associated nicking activity followed by the action of a 3'-repair diesterase which cleaves 5' to the incised AP site. The answer to the question of whether these two proposed pathways operate in vivo and what types of damage each might repair awaits the isolation of a mutant deficient in dRpase. Regardless of the pathway used, AP endonucleases are essential for the base-excision repair of D N A and will be discussed in depth in this review. T h e c h e m i s t r y of A P sites

An AP site in D N A is a mixture of chemical species in equilibrium between an open-chain aldehyde, an open*chain hydrate, and cyclic hemiacetals (Fig. 2). Characterization of abasic sites in duplex D N A has shown that the two anomers of the hemiacetal are found in equal amounts and constitute the vast majority of species (Manoharan

5' o

"O-pI ~0 I o

I

y'

5'

o:1

O "o-~ =o I

3"

5'

g

.o-, =o

=o

!

I

c

A

o

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~

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~

O I 3'

,.

C

O _-o

I 5' t

=o

3'

?

"O-p =0 I

o "o-~ =o ! O 1 3'

Fig. 2. The chemical structure of an AP site. An AP site can exist as (A) the open chain aldehyde, as (B and C) the a- and fl-hemiaeetals, and as (D) the open-chain hydrate.

et al., 1988b). Open-chain aldehydes constitute about 1% of the total AP sites (Wilde et al., 1989), but they are the predominant chemical species in terms of reactivity. Since an AP site can be found opposite 4 different bases, all AP sites are not chemically equivalent and, in fact, 4 distinct AP sites exist in D N A . AP sites m a y undergo several chemical reactions (Fig. 3). Phosphodiester b o n d cleavage adjacent to an AP site can occur via a fl-elimination reaction catalyzed by nucleophiles. This reaction may proceed through two pathways. One p a t h is initiated by the removal of a proton from the C H 2 group of the deoxyribose a to the carbonyl group at C-1. The o t h e r path involves the formation of a Schiff base between an amine and the C-1 carbonyl group of the ring open aldehyde. Both of these events are followed by a fl-elimination reaction. In both cases, the group left at the 3'-terminus is an

176 5'

~ O I

5.

A

D

O I

" O - P ~---0

I

"O-P = 0

0

I

I

0

o

I

H:zC. OHI~I

C----CH

o=c / (~

I

~CH2~/H2

H "e-- :B

"O-I ~ .=O I

O k

"O

5'

3'

~ O

"O-I ~ ~ O

C

t

O

O-p = O I

o

OH

3'

I

"o 5g

O

,

B

t

?

I

3'

"O-p = 0 0 H2C

5' t O

excess

O"

~g__~'"

E

O-P =O I

O

OH CH2

~

CH2

H

' (°.o

"O-~ =O I

O

3'

"o "o-~ ~-~o I

o k 3' Fig. 3. Chemical reactions occurring at an AP site. An AP site (A) can undergo a fl-elimination (B) to yield phosphodiester bond cleavage. The 3'-terminus of the nick (C) is an a,fl-unsaturated aldehyde, ~hydroxy-2-pentenal. The ct,fl-unsaturated aldehyde can rearrange in alkaline solution to yield a 3'-2-oxocyclopent-l-enyl terminus (D). The unsaturated aldehyde can also undergo a 8-elimination to yield a one nucleotide gap in the D N A bordered by a 5'-phosphoryl and a 3'-phosphoryl terminus and free 4-hydroxy-pent-2,4-dienal.

a, fl-unsaturated aldehyde, 4-hydroxy-2-pentenal, and the 5'-terminus is a C-5 phosphorylated sugar. The 4-hydroxy-2-pentenal terminus can exist as a trans aldehyde or as a cis aldehyde in equilibrium with the hemiacetals. Under conditions of excess catalyst, a second elimination, a 8-elimination, can occur which yields a C-3 phosphorylated sugar at the 3'-terminus and the release of 4-hydroxy-pent2,4-dienal. Under alkaline conditions, the 4-hydroxy-2-pentenal terminus can rearrange to form

a 3'-2-oxocyclopent-l-enyl terminus (Jones et al., 1968). Other reactions can occur at AP sites in alkaline solution. Cleavage 3' to an AP site can occur when the hydroxyl group at C-4 of the ring open sugar participates in a transphosphorylation to yield a 3',4'-cyclization and cleavage 3' to the AP site ( T a m m et al., 1953). A 4',5'-cyclization can result in cleavage 5' to the AP site (Bayley et al., 1961). These cyclization reactions should yield

177

dephosphorylated D N A fragments. Such dephosphorylated fragments are not found in large quantities after alkaline hydrolysis (Lindahl and Andersson, 1972) suggesting that fl-elimination is the major pathway for the cleavage of the phosphodiester backbone at AP sites. The a,fl-unsaturated aldehyde found after a fl-elimination is quite reactive. Nucleophiles add to a,fl-unsaturated carbonyl compounds quite readily. Manoharan et al. (1988b) and Bailly and Vedy (1988a) have reported that thiols react readily with the unsaturated aldehyde produced by a B-elimination reaction. This addition reaction can compete with the 8-elimination reaction mentioned above (BaiUy and Verly, 1988). The ~t,flunsaturated carbonyl compounds can also participate in electrophilic addition reactions. The structure of AP sites

The structure of D N A containing AP sites has been determined in several laboratories. The general approach has been to examine synthetic oligonucleotides containing a base-free analog of 2-deoxyribose by N M R spectroscopy. Cuniasse et al. (1987) used 1,4-anhydro-2-deoxy-D-ribitol to mimic an AP site in DNA. They placed an adenine opposite this sugar to create an apyrimidinic site. The adenine opposite the AP site lies within the D N A helix and adopts the anti conformation. In addition, they found that the oligonucleotide containing the AP site retains all the characteristics of the classical B D N A structure. Rapp et al. (1987) used an oligonucleotide in which methyl 2'-deoxya/fl-D-ribofuranoside mimics an AP site. A cytosine was placed opposite this sugar to create an apurinic site. The repeating nature of the sequence of this oligonucleotide resulted in a structure in which the abasic sugar was not in classical Watson-Crick DNA. Kalnik et al. (1988) synthesized an oligonucleotide in which 3-hydroxy-2-(hydroxymethyl)tetrahydrofuran mimics an apyrimidinic site with an adenine in the complementary strand. Their data suggest that the adenine opposite the AP site lies within the helix and stacks with the base pairs on both sides of the site. All the bases are found in the anti conformation and the DNA is right-handed throughout the oligonucleotide. Two backbone phosphates appear to

have altered conformations; however, the exact phosphates were not identified. Further studies with acyclic abasic sites (Kalnik et al., 1989) showed that the abasic sites are structurally similar if the analog of 2-deoxyribose is not a 5-membered closed ring and if the backbone is shortened by one carbon residue. The locations of distorted backbone phosphodiester bonds were found to be on either side of the AP site. In addition, a phosphodiester bond 3' to the deoxyadenosine opposite the AP site was distorted. In summary, the structure of AP sites has been determined for several synthetic oligonucleotides containing modified AP sites. In the two instances where the AP sites were found in duplex DNA, the base opposite the AP site (adenine in both instances) was found stacked into the helix and the DNA retained most of the features of classical B-DNA. These studies all replaced 2-deoxyribose with reduced sugars to prevent ring opening and possible fl-elimination at the AP site. Manoharan et al. (1988a) have shown that the conformational features of a true AP site depend upon the opposing base and also that the deoxyribose at the AP site shows different equilibrium distributions of aldehyde and hemiacetals depending upon the opposing base. There will probably be a family of structures for AP sites depending upon the opposing base and also possibly depending upon the surrounding sequence. The flexibility of the structure of AP sites will become clearer when more model oligonucleotides are studied. Model systems for AP endonucleases

Tripeptides containing aromatic and basic amino acids have been shown to bind to D N A containing AP sites (Behmoaras et al., 1981a; H616ne et al., 1982) and catalyze the cleavage of the phosphodiester bond adjacent to the AP site (Behmoaras et al., 1981b; Pierre and Laval, 1981; Duker and Hart, 1982; H616ne et al., 1982). The tripeptide lysyltryptophyl-a-lysine ( L y s - T r p - L y s ) has been most thoroughly studied. Behmoaras et al. (1981a) showed that L y s - T r p - L y s b o u n d tightly to D N A containing apurinic sites. The binding was also specific, showing a 200-fold preference for D N A containing AP sites over native DNA. A two-step mechanism for the binding of

178 the tripeptide was proposed. The first step in binding involves only electrostatic interactions between the lysine residues and the phosphates on the D N A backbone. The second step involves the stacking of the tryptophan into the D N A helix at the abasic site. Further studies (Pierre and Laval, 1981) showed that the tripeptide could catalyze the cleavage of a phosphodiester bond adjacent to the AP site. The nicking was much reduced when the tripeptides L y s - A l a - L y s or L y s - L y s - L y s were used suggesting that the stacking of an aromatic residue into the helix was required for nicking. The nicking by the tripeptide was ascribed to a fl-elimination reaction and it was shown that reduction of the AP site with sodium borohydride suppressed nicking as would be expected if the open-chain aldehyde form of the deoxyribose was the reactive species. Two mechanisms can account for the flelimination reaction at the AP site by the tripeptide L y s - T r p - L y s . The tight and specific binding of the tripeptide at AP sites will place a lysine residue in close proximity to the base-free deoxyribose. Either a proton abstraction at the C-2 carbon of deoxyribose or the formation of a Schiff base will result in a fl-elimination reaction. Hrlrne et al. (1982) showed that sodium cyanoborohydride could trap the tripeptide covalently linked to DNA as expected if a Schiff base between a lysine and the aldehyde of a ring-open deoxyribose were formed and then reduced. Since only a portion of the tripeptide could be covalently attached to DNA, the possibility that part of the reaction proceeded by proton abstraction could not be eliminated. The exact nature of the cleavage of the phosphodiester bond at an AP site was not fully understood although previous work (Livingston, 1964; Coombs and Livingston, 1969) had suggested a pathway involving a Schiff base and Hrlrne's work with the tripeptide L y s - T r p - L y s also suggested a Schiff base intermediate. Recent work has shown that methoxyamine does form a Schiff base with an AP site in DNA (Vasseur et al., 1986). Additionally, the intercalating agent 9-aminoellipticine is able to cleave AP sites at high efficiency and has been shown to form a Schiff base in the course of the cleavage reaction (Bertrand et al., 1989).

The model systems utilizing tripeptides suggest several features for the recognition and binding of AP sites by AP endonucleases and for the reaction mechanism for one type of cleavage event. AP endonueleases from prokaryotes The AP endonucleases of prokaryotic origin can be divided into two groups, the class I AP endonucleases which cleave 3' to AP sites and the class II AP endonucleases which cleave 5' to AP sites (Mossbaugh and Linn, 1980). Verly and colleagues have found that the cleavage carried out by one class I AP endonuclease appeared to be a fl-elimination reaction (Fig. 1) and suggested that such enzymes be called fl-elimination catalysts (Bailly and Verly, 1987). Kim and Linn (1988) found evidence suggesting that class I AP endonucleases worked by a fl-elimination reaction as well but suggested that although a hydrolytic mechanism was not involved, class I AP endonucleases could still be viewed as lyase-type phosphodiesterases. Bailly and Verly (1989b) have more recently suggested that AP site-DNA 5'-phosphomonoester lyase or AP lyase is the appropriate systematic name for this class of enzyme. In summary, class I AP endonucleases catalyze a fl-elimination reaction at AP sites and should be called AP lyases. Class II AP endonucleases cleave phosphodiester bonds hydrolytically and are nucleotidyl hydrolases. For convenience, we will use the terminology AP endonuclease for enzymes originally placed in class II and AP lyase for enzymes originally placed in class I. Additional changes in nomenclature may be anticipated since all well characterized AP lyases also have associated D N A N-glycosylase activities which may in some cases be mechanistically coupled to the AP lyase activity. Exonuclease III. Exonuclease III was first identified as a 3'-5'-exonuclease which degraded double-stranded D N A (Richardson et al., 1961) and as a 3'-phosphatase (Richardson and Kornberg, 1961). These studies and results reported by Roychoudhury and Wu (1977) showed that exonuclease III degraded D N A containing ribonucleotides and would remove a terminal 3'-phosphate from a polynucleotide terminated by a 3'-

179 phosphorylribonucleoside. Exonuclease III was subsequently shown to have RNAase H activity by two laboratories (Keller and Crouch, 1972; Weiss et al., 1978). Keller and Crouch found that exonuclease III could degrade poly(rA), poly(dT) to 5'-ribomononucleotides and 5'-deoxyribonucleotides. The studies by Weiss and colleagues showed that the R N A strand of a D N A . R N A hybrid was degraded at 10000 times the rate of the D N A strand using a highly purified enzyme preparation. The discovery that exonuclease III had an AP endonuclease activity occurred by chance when it was discovered that a collection of endonuclease II mutants isolated by Yajko and Weiss (1975) were also all deficient in exonuclease III. Upon further investigation, it was shown that a collection of exonuclease III mutants isolated by Milcarek and Weiss (1972) were also all deficient in endonuclease II. A number of biochemical and genetic experiments (Weiss, 1976; White et al., 1976; Rogers and Weiss, 1980a) showed that the mutations affecting both exonuclease III and endonuclease II were cotransducible and corevertible, that both activities were coded for by a cloned fragment of the E. coli chromosome 3 kb in length, and that a single purified peptide had both exonuclease and endonuclease activity. The 3'-phosphatase activity described by Richardson and Kornberg (1961) was also shown to be an activity of exonuclease III by the criteria described above. Other AP endonuclease activities have been described (see Weiss, 1981 for a review) and these activities, endonuclease II (Friedberg and Goldthwait, 1969) and endonuclease VI (Verly and Rassart, 1975; Gossard and Verly, 1978) are now known to be the AP endonuclease activity of exonuclease III. Weiss (1976, 1981) proposed a model to explain how a small protein of approx. 30000 molecular weight could catalyze the 4 known reactions ascribed to exonuclease III; the 3'-phosphatase activity, the RNAase H activity, the 3'-5'-exonuclease activity and the AP endonuclease activity. He suggested that exonuclease III had a single catalytic site which would carry out the hydrolysis of either the phosphodiester or the phosphomonoester bond required in the 4 reactions. In addition to a catalytic site, he proposed that 2

other regions of the protein were necessary to recognize duplex D N A and discriminate between the RNA strand and the D N A strand in a D N A . R N A hybrid and to recognize the feature found in all the substrates, a space created by a missing or a displaced base. Weiss hypothesized that the fraying which occurred at the terminus of a D N A strand in duplex D N A would look like a missing base. Two more activities of exonuclease III have been recently reported. Exonuclease III has a 3'phosphodiesterase activity which removes 3'-phosphoglycolate residues (Niwa and Moses, 1981; Henner et al., 1983; Demple et al., 1986) and 3'-phosphoglycolaldehyde esters (Demple et al., 1986). This activity also may recognize the 3'-a, flunsaturated aldehyde residue left after a r elimination reaction since exonuclease III treatment can activate endonuclease III-incised D N A to be a substrate for D N A polymerase I (Warner et al., 1980; Mosbaugh and Linn, 1982). In addition, Kow and Wallace (1985) have shown that exonuclease III incises D N A 5' to urea residues. This u r e a - D N A endonuclease activity catalyzes a reaction very similar to the previously described reaction at AP sites. Kow (1989) has examined this activity in more detail. Exonuclease III will cleave the phosphodiester bond next to O-alkylhydroxylamine N-glycosides as well as next to urea N-glycosides in DNA. Thymine glycol N-glycoside, dihydrothymine N-glycoside and formamidopyrimidine N-glycoside do not serve as substrates. All the residues which are substrates have a secondary amine at the N-glycosyl bond while those that are not substrates have a tertiary amine at this position. A single exception to this pattern is formamidopyrimidine N-glycoside which has a secondary amine at the N-glycosyl bond, yet is not a substrate. Kow has suggested that there may be two requirements for recognition and cleavage; a secondary amine at the N-glycosyl bond and lack of base pairing by the damaged base. Kow (1989) has proposed that the ring opening of the sugar attached to the damaged base would be facilitated by a secondary amine at the N-glycosyl bond. This model suggests that ring opening leads to an imine bond between the C-1 of the sugar and the N-1 of the base with the concom-

180 itant movement of the base out of the D N A helix. The rotation of the base out of the helix would also be facilitated by lack of base pairing with a base in the complementary strand. This space in the D N A helix and the space found at true AP sites are the features of the substrate that allow exonuclease III to bind and hydrolyze the phosphodiester bond 5' to the lesion. If this space is an important part of the substrate, and if the Weiss model is correct, then the model for the action of exonuclease III at the terminus of a D N A strand needs to be modified. In fact, Kow (1989) has suggested that the structure recognized at the 3'terminus of duplex D N A is not merely a frayed end, but a ring-opened nucleotide. The ring opening of the sugar would lead to the formation of an unstable iminium bond due to the presence of the tertiary amine at the N-glycosyl bond. Exonuclease III would stabilize this iminium intermediate so that the base could rotate out of the helix to generate a space 5' to the terminal base. This substrate is the equivalent of the substrate recognized by the AP endonuclease activity of exonuclease III. The proposed mechanism (Kow, 1989) unites the exonuclease and endonuclease activities of exonuclease III by modifying the substrate recognized in the Weiss model. Ring-opened sugars allow the rotation of a 3'-terminal base or an interior base with a secondary amine at the N-glycosyl bond to create a space 5' to the target nucleoside. It is worth noting that the hydroxyl group at C-2 of ribose in R N A would hinder the ring opening (Kochetkov and Budovskii, 1972) which is proposed as the first step in enzymatic action. The expectation that R N A hydrolysis would be slower than D N A hydrolysis is fulfilled by rate measurements which show the relative rate of phosphodiester bond cleavage by exonuclease III is 8-fold higher for the exodeoxyribonuclease compared to the RNAase H activity (Rogers and Weiss, 1980b). In the case of the phosp h o m o n o e s t e r a s e activity a n d the p h o s phodiesterase activity at phosphoglycolates, a space would preexist at the site of cleavage since there is no base 3' to the site. Exonuclease III requires Mg 2÷ or Mn 2÷ for o p t i m u m activity. The purified enzyme requires no added divalent cations in the absence of chelat-

ing agents, but is inactive in the presence of E D T A suggesting that the purified enzyme has a bound metal ion (Weiss, 1981). Ca 2÷ can substitute for Mg 2÷ for AP endonuclease activity and can partially substitute for Mg 2+ for other activities (Kow, 1989). The mode by which exonuclease III degrades D N A exonucleolytically is determined by temperature. At 5°C in 70 m M NaC1, exonuclease III removes 6 nucleotides from the end of a duplex and remains bound to the D N A in a stable complex (Donelson and Wu, 1972). At lower salt concentrations, there is a slower rate of hydrolysis after the initial burst which releases 6 nucleotides. If the stable e n z y m e - D N A complex formed in high salt is subjected to a temperature shift, hydrolysis resumes at 2 3 ° C or 37°C. Hydrolysis ceases if the temperature is returned to 5°C. At 2 3 - 2 8 ° C digestion is synchronous ( + 5%) for 250 nucleotides (Wu et al., 1976). Exonuclease III degrades at least 100 nucleotides in a processive fashion at 23°C (Wu et al., 1976), yet it acts in a distributive fashion at 37°C (Thomas and Olivera, 1978). An Arrhenius plot of the temperature dependence of the exonucleolytic rate of exonuclease III shows a transition at 25°C (Kow, 1989) or at 30°C (Henikoff, 1987). This transition is seen in the presence of Mg 2+ but not in the presence of Ca 2÷ (Kow, 1989). The activation energy in the presence of Ca 2÷ is similar to the activation energy in the presence of Mg 2+ at low temperatures and is higher than the activation energy in the presence of Mg 2÷ at high temperature. Kow (1989) has suggested that exonuclease III exists in two conformers, one which binds tightly to D N A below 25°C and is processive on D N A and one which is distributive above 25°C. The higher activation energy seen at temperatures below 25°C may reflect the energy required for the movement of the enzyme on D N A while the lower activation energy seen at higher temperatures may reflect the energy required for hydrolysis. Since only the high-energy conformer is seen in the presence of Ca 2÷, it may be that Ca 2÷ holds exonuclease III in the processive or tightly binding conformer (Kow, 1989). Endonuclease IV. Endonuclease IV was found when mutants deficient in exonuclease III were

181 examined carefully and found to have a residual AP endonuclease activity (Ljungquist et al., 1976) which was separable from exonuclease III by chromatographic procedures (Ljungquist and Lindahl, 1977). The enzyme was partially purified and characterized as an enzyme which cleaved D N A containing AP sites (Ljungquist, 1977). The gene coding for endonuclease IV, the nfo gene, has been cloned (Cunningham et al., 1986) and subsequently it was shown that endonuclease IV is inducible by treatment with paraquat (Chan and Weiss, 1987). A 200-fold overproduction of endonuclease IV can be obtained by inducing cells carrying a recombinant plasmid containing the nfo gene (Chan and Weiss, 1987). Levin et al. (1988) have purified endonuclease IV to homogeneity using such overproducing cells as a source of enzyme. As had been anticipated from previous experiments (Demple et al., 1986; Mosbaugh and Linn, 1982), endonuclease IV will remove phosphoglycolaldehyde, phosphate, deoxyribose-5phosphate and the 4-hydroxy-2-pentenal residue from the 3'-terminus of duplex DNA. Bailly and Verly (1989a) have unambiguously shown that endonuclease IV cleaves 5' to the AP site leaving a 3'-hydroxyl group. Endonuclease IV can be inactivated by EDTA in the presence of substrate suggesting that a tightly bound essential metal ion is present in the purified protein. Mn 2+ can partially restore activity to inactivated enzyme, but the nature of the metal cofactor remains to be determined (Levin et al., 1988). Endonuclease IV has been shown to have several activities in common with exonuclease III; (i) an AP endonuclease activity which cleaves 5' to AP sites, (ii) a phosphomonoesterase activity which removes 3'-phosphoryl groups, (iii) a phosphodiesterase activity which removes 3'-phosphoglycolates and other blocked 3'-termini. In addition, it has been reported that endonuclease IV has a u r e a - D N A endonuclease activity as well (Wallace et al., 1988). The major difference between exonuclease III and endonuclease IV is the absence of an exonuclease activity in endonuclease IV. Despite the similarity in size and in the reactions catalyzed, exonuclease III and endonuclease IV show no significant similarity at the level of protein primary sequence (Saporito et al., 1988; Saporito and Cunningham, 1988).

Endonuclease IlL Endonuclease III was originally described as an endonuclease that nicked ultraviolet irradiated D N A (Radman, 1976). Gates and Linn (1977a) subsequently described an enzyme which appeared to be the same as the one described by Radman. This enzyme acted upon D N A damaged by osmium tetroxide, acid or Xrays as well as UV-irradiation. Demple and Linn (1980) showed that endonuclease III could catalyze the release of 5,6-dihydroxydihydrothymine and 5,6-dihydrothymine from D N A and suggested that endonuclease III had both a D N A N-glycosylase activity and a class I AP endonuclease activity. Katcher and Wallace (1983) showed that endonuclease III was the X-ray endonuclease activity that had been partially purified by Wallace and coworkers (Strniste and Wallace, 1975; Armel et al., 1978). In addition, they showed that endonuclease III had a D N A N-glycosylase activity which recognized urea residues and thymine glycol residues. Breimer and Lindahl (1984) found that endonuclease III was the same enzyme as the u r e a - D N A glycosylase they had previously described (Breimer and Lindahl, 1980) and also showed that the glycosylase activity would remove thymine, glycol, urea, 5-hydroxy-5-methylhydantoin and methyltartronylurea from DNA. Breimer and Lindahl (1985) showed that the Nglycosylase activity of endonuclease III could release both cis- and trans-thymine glycol, 6-hydroxy-5,6-dihydrothymine, and pyruvylurea from DNA. Endonuclease III cleaves at the site of cytosine residues in UV-damaged D N A (Doetsch et al., 1986; Helland et al., 1986; Weiss and Duker, 1986; Weiss and Duker, 1987). Boorstein et al. (1989) have shown that endonuclease III releases cytosine photohydrate from D N A as well as uracil photohydrate. Finally, it has been noticed that endonuclease III incises damaged D N A at guanine residues in D N A damaged by oxidizing agents (Helland et al., 1986; Gossett et al., 1988). The damaged guanine residue recognized by endonuclease III has not as yet been identified. Endonuclease III requires no divalent cations and is EDTA-resistant (Radman, 1976; Gates and Linn, 1977). The gene for endonuclease III, the nth gene, has been cloned (Cunningham and Weiss, 1985) and sequenced (Asahara et al., 1989). The cloned

182 gene has been inserted into an expression vector which yields a 300-fold overproduction of enzyme and which has allowed for the purification of large amounts of the protein to homogeneity (Asahara et al., 1988). The protein has a predicted molecular weight of 23 546, is basic, behaves as a monomer in solution and has a sedimentation coefficient of 2.65 S in agreement with the properties of endonuclease III characterized in the various laboratories mentioned above. Purification of large amounts of endonuclease III led to an unexpected finding. Purified endonuclease III has a chromophore which absorbs at 410 nm (Asahara et al., 1989). Elemental analysis, M~Sssbauer spectroscopy and EPR analysis all showed that endonuclease III is an iron-sulfur protein (Cunningham et al., 1989). The data obtained from MiSssbauer spectroscopy are typical of a 4 F e - 4 S cluster in the 2 + core oxidation state. The 4 iron subsites of the cluster appear to be found in similar environments suggesting that there is a homogeneous ligand structure within endonuclease III, with cysteines being the likely ligands. The presence of a 4 F e - 4 S cluster in endonuclease III is a unique occurrence in the known DNA-repair enzymes, and the possible role of the cluster in enzyme action remains unknown. Understanding of the mechanism of action of endonuclease III has been slow, partially due to an unexpected reaction mechanism for the cleavage of the phosphodiester bond at AP sites. Originally, it was proposed that class I AP endonucleases caused the cleavage of a phosphodiester bond 3' to an AP site leaving a 3'-deoxyribose (Warner et al., 1980; Mosbaugh and Linn, 1982). The signature of this 3'-deoxyribose was the inabihty of E. coli D N A polymerase I to use this terminus as a primer. Subsequently, Bailly and Verly (1984) showed that D N A polymerase I would use a substrate with an authentic 3'-deoxyribose quite efficiently, and they suggested that the class I AP endonucleases did not cleave by hydrolysis, but rather that they left a 3'-terminus that was not a 3'-deoxyribose. Bailly and Verly (1987) presented data suggesting that endonuclease III cleaved the phosphodiester bond 3' to an AP site by a r-elimination reaction. They found that cleavage with endonuclease I I I resulted in a characteristic doublet when an oligonucleo-

tide containing a unique AP site was treated with endonuclease III and displayed on a D N A sequencing gel. One of the bands is the primary product of the r-elimination reaction, and the other is a product of a non-enzymatic side reaction (Bricteux-Grrgoire and Verly, 1989). In addition, they found that 3H at the C-2 position of the sugar was lost in the course of the reaction as would be expected for a r-elimination reaction. K i m and Linn (1988) also suggested that endonuclease III catalyzed a B-elimination reaction based on the chromatographic analysis of the sugar residue released by endonuclease III followed by alkaline treatment. Manoharan et al. (1988b) have directly proven that the cleavage 3' to an AP site by T4 endonuclease V occurs by a r-elimination reaction. They used 13C N M R analysis to show that the product of the reaction is the expected a,fl-unsaturated aldehyde. Mazumder et al. (1989) in further studies have reported the stereochemical course of the reaction. Using stereospecifically labeled AP sites, they showed that the enzyme abstracts the pro-S 2-hydrogen. ~H N M R spectroscopy was used to show that the a,fl-unsaturated aldehyde has trans geometry. They concluded from these studies that the stereochemistry of the r-elimination reaction is syn, and that the reaction proceeds from an open-chain form of the abasic site; the substrate m a y be the acyclic aldehyde found at abasic sites, or it may be an activated form of the aldehyde such as an imine. Endonuclease III behaves exactly like endonuclease V in similar studies (A. Mazumder, J.A. Gerlt, M.J. Absalon, J. Stubbe, R.P. Cunningham, J.A. Withka and P.H. Bolton, unpublished observations) and catalyzes a r-elimination reaction following the same stereochemical course. Kow and Wallace (1987) have suggested that the N-glycosylase activity and the AP lyase activity of endonuclease III work in a concerted fashion. They could not find unnicked AP sites in D N A when the substrate for the reaction was D N A containing thymine glycol or urea. Futhermore, they found that magnesium would inhibit the AP lyase activity of endonuclease I I I at AP sites, but had no effect on the nicking activity at thymine glycol residues. They suggested a reaction mechanism in which both the N-glycosylase activity and the AP lyase activity are associated in a

183 common reaction pathway. The first step in the pathway is the protonation of the ring oxygen of the deoxyribose followed by the formation of a Schiff base between the sugar and the base. This is similar to one of the pathways proposed for the acid hydrolysis of nucleosides (Kenner, 1957; Dekker, 1960; Cadet and Teoule, 1974). The next step in the reaction pathway is a transiminization reaction in which a Schiff base is formed between the enzyme and the C-1 aldehyde of the sugar with the release of the damaged base. The Schiff base between the sugar and the base would lead to the fl-elimination of the 3'-phosphate. Their model predicts that the rate of the concerted reaction (cleavage of the phosphodiester bond adjacent to a damaged base) will be greater than for cleavage at AP sites since the transiminization reaction between the enzyme and the ring opened form of the damaged nucleoside would proceed more rapidly than a Schiff base formation between the ring-opened aldehyde and the enzyme at an AP site. This prediction and others made upon the assumption of their reaction pathway have been fulfilled experimentally (Kow and Wallace, 1987). This elegant model conforms to known reaction pathways and should surely stimulate further studies to confirm the proposed reaction mechanism. Formamidopyrimidine-DNA glycosylase. Formamidopyrimidine-DNA glycosylase ( f a p y - D N A glycosylase) has an associated AP lyase activity (O'Connor and Laval, 1989). F a p y - D N A glycosylase was originally identified by Chetsanga and Lindahl (1979) as an activity which released 2,6diamino-4-hydroxy-5N-methylformamidopyrimidine residues from DNA. The N-glycosylase activity required no divalent cations and was not inhibited by EDTA. Breimer (1984) later showed that the enzyme also released 4,6-diamino-5-formamidopyrimidine from DNA. Boiteux et al. (1987) cloned the gene for f a p y - D N A glycosylase, the fpg gene, and overproduced the protein. The predicted amino acid sequence for f a p y - D N A glycosylase yields a protein with a calculated molecular mass of 30.2 kD (Boiteux et al., 1987). O'Connor and Laval (1989) found that an AP nicking activity and f a p y - D N A glycosylase coeluted in several different chromatographic steps during the purification of the protein to electro-

phoretic homogeneity. Since the nicking activity would not cleave reduced AP sites, they suggested that the activity was an AP lyase. The AP lyase activity is EDTA resistant. They also found that the enzyme apparently left both 5'-phosphoryl and 3'-phosphoryl end groups in contrast to the previously described endonuclease III which left a 3'-a, fl-unsaturated aldehyde end group. Bailly et al. (1989c) examined the nicking activity of f a p y D N A glycosylase in more detail and found that the enzyme appears to catalyze a fl,g-elimination at AP sites. The presence of 2-mercaptoethanol does not block the g-elimination. If the enzyme were to dissociate from the unsaturated aldehyde after the fl-elimination, the nucleophilic 2mercaptoethanol would react with the a, fl-unsaturated aldehyde and prevent the g-elimination (Bailly and Verly, 1988a). It has not been determined if the N-glycosylase activity and the fl-elimination activity work in a concerted fashion for f a p y - D N A glycosylase and, therefore, it is not known if f a p y - D N A glycosylase and endonuclease III share a common reaction mechanism. The enzymes do differ in their ability to carry out the g-elimination step of the AP lyase activity. T4 and M. luteus U V endonucleases. Both bacteriophage T4 and Micrococcus luteus have UV endonucleases that are also pyrimidine d i m e r D N A glycosylases (Gordon and Haseltine, 1980; Haseltine et al., 1980; Radany and Friedberg, 1980; Seawell et al., 1980; McMillan et al., 1981; Warner et al., 1981). The nicking activities and the N-glycosylase activities have been shown to be physically associated (Nakabeppu and Sekiguchi, 1981; Grafstrom et al., 1982). Recently, it has been demonstrated that the T4 enzyme cleaves DNA by a B-elimination reaction (Manoharan et al. 1988b; Mazumder et al., 1989) while Bailly et al. (1989b) have proposed that both enzymes cleave by a fl,g-elimination. The enzymes are of similar molecular weight and share antigenic determinants (Yarosh and Cecolli, 1989) and may be related phylogenetically. Since the T4 enzyme has been more extensively studied, we will concentrate on the results obtained with this enzyme. This enzyme is EDTA-resistant and is only slightly stimulated by Mg z+ or Mn 2+ (Friedberg and King, 1969; Yasuda and Sekiguchi, 1970).

184

Several fines of evidence suggest that the N-glycosylase activity and the AP lyase activity of T4 endonuclease V are not coupled. Nakabeppu and Sekiguchi (1981) have shown that during the course of a reaction using D N A containing pyrimidine dimers as substrate, the rate of formation of AP sites exceeds the rate of formation of nicks. The same authors also showed that the two activities exhibited different thermosensitivities and different p H optima. Bonura et al. (1982a) showed that the presence of competing D N A in a reaction mixture only inhibited the AP lyase activity and suggested that endonuclease V could dissociate from D N A after cleavage of the N-glycosyl bond but prior to cleavage of the phosphodiester bond. Nakabeppu et al. (1982) have purified mutant forms of endonuclease V and shown that one of the enzymes retained the N-glycosylase activity but had lost the AP lyase activity. These results all support the notion that the two activities are not coupled and pose the question as to what reaction mechanisms can be considered for endonuclease V action. The acid hydrolysis of nucleosides can occur by a mechanism different from that proposed for endonuclease III action. This second mechanism (Shapiro and Kang, 1969; Zoltewicz et al., 1970; Garrett and Mehta, 1972) starts with the protonation of the base to form a monocation or dication which leads to the hydrolysis of the N-glycosyl bond. This fragmentation of the nucleoside leads to the formation of a free base and a cyclic carbonium form of deoxyribose. The carboxonium ion is converted to deoxyribose upon the addition of H20. This mechanism would allow for cleavage of the N-glycosyl bond without the concerted cleavage of the phosphodiester bond. Dodson and Lloyd (1989) have proposed a similar mechanism for the N-glycosylase activity of T4 endonuclease V. They propose that the protonation of the carbonyl group at C-2 in one of the pyrimidines of a dimer is followed by rupture of the N-glycosyl bond to yield a carbocation form of deoxyribose which could be converted to deoxyribose by reaction with hydroxide ion. The enzyme could dissociate at this point or it could remain bound and catalyze a fl-elimination or a fl,8-elimination. The elimination reaction could be catalyzed by either Schiff base formation or a proton abstraction. A

different model has been proposed (Weiss and Grossman, 1987) in which a nucleophilic attack by T4 endonuclease V on the 5'-N-glycosyl bond of the dimer forms a Schiff base with the concomitant rupture of the N-glycosyl bond. A fl-elimination follows to give strand cleavage. This model is somewhat like that proposed by Kow (1989) for endonuclease III and might imply a concerted reaction mechanism. It is obviously of great interest to determine if the N-glycosylase activities of endonuclease III and T4 endonuclease V utilize different reaction mechanisms. It will also be of interest to see which mechanism f a p y - D N A glycosylase uses since it has been suggested that T4 endonuclease V and f a p y - D N A glycosylase share the ability to carry out a fl,8-elimination. There may be two quite distinct classes of AP lyases based upon reaction mechanisms. Finally, it is also of great interest to determine the mechanism of action of a D N A N-glycosylase such as u r a c i l - D N A glycosylase that does not have an associated AP lyase to see how an elimination reaction at the AP site is avoided. We will not discuss other aspects of T4 endonuclease V since they have been reviewed in depth very recently by Dodson and Lloyd (1989).

Endonuclease V. An enzyme found in uninfected E. coli which recognizes and cleaves AP sites was described by Gates and Linn (1977b) and Demple and Linn (1982). This enzyme was called endonuclease V (not to be confused with T4 endonuclease V) and was shown to be distinct from the other known AP endonucleases. It is active on single- and double-stranded D N A containing uracil or AP sites or on duplex D N A treated with OsO 4 or UV light. Endonuclease V requires Mg 2+ for activity and Ca 2+ can partially substitute for Mg 2+ (Gates and Linn, 1977b). Bernelot-Moens and Demple (1989) have suggested that endonuclease V may be identical to one of the D N A 3'-repair diesterases they have identified in E. coli. This finding suggests some similarities between exonuclease III, endonuclease IV and endonuclease V in that they all can cleave at AP sites, that they can cleave endonucleolytically at base residues (urea or uracil) and that they have diesterase activities on 3'-blocked termini.

185 Endonuclease VII. Endonuclease VII, also called single-stranded AP endonuclease cleaves single-stranded D N A containing apyrimidinic sites (Bonura et al., 1982b). The enzyme is specific for single-stranded DNA, is active in the presence of E D T A and will not cleave D N A containing reduced AP sites. Bonura et al. (1982b) suggest that the enzyme catalyzes a fl-elimination reaction but, unfortunately, the termini left after cleavage were not characterized to help confirm this proposal.

AP endonucleases from lower eukaryotes AP endonucleases have been identified and characterized in a wide variety of eukaryotes and extensively studied in yeast and Drosophila. The physical properties, reaction parameters and substrate specificities of these enzymes differ greatly amongst the species where they have been found. In addition, multiple forms of AP endonucleases have been found in single Organisms and within the same cell type. The present discussion of eukaryotic AP endonucleases will be limited primarily to recent studies in organisms in which these enzymes have been extensively studied. For a summary of earlier studie~ including those on algae and plants, the reader is referred to Wallace (1988). S. cerevisiae A P endonucleases. A relatively large number of AP endonucleases have been isolated from yeast. The earlier studies (Pinon, 1970; Chiebowicz and Jachymczyk, 1977; Bryant and Haynes, 1978; Futcher and Morgan, 1979) focused on the identification of nicking activities directed against various depurinated D N A substrates. It was also observed that the levels of such AP endonuclease activities were about the same in both wild-type and in 25 D N A repair-deficient (rad, rev, m m s and mut mutant) strains (Chiebowicz and Jachymczyk, 1977; Futcher and Morgan, 1979). Two AP endonucleases, A and B, that possessed broadly similar properties with respect to size (24 kDa) and cofactor requirements (resistant to EDTA, but stimulated by Mg 2+) but which differed in their pH optima, heat sensitivity and inhibition by p-chloromercuribenzoate were purified by Akhmedov et al. (1982). The location of strand cleavage (3' or 5') to the AP site and the

nature of the resulting termini following such cleavage were not determined for either of these endonucleases. A yeast mitochondrial AP endonuclease has also been isolated from the inner mitochondrial membrane (Foury, 1982). Armel and Wallace (1978, 1984) purified 5 chromatographically distinct AP endonucleases, D1, D2, D3, D4 and E that ranged in size from 10 to 49 kDa. All 5 activities apparently cleaved depurinated substrates 5' to the AP site, leaving a 3'-terminal hydroxyl group that was a substrate for DNA polymerase. The 5 activities differed somewhat with respect to p H optima, salt and Mg 2+ concentration effects. Endonucleases D4 and E also nicked OsO4-oxidized D N A but only at a fraction of the extent of nicking observed for E. coli endonuclease III on the same substrate. Chang et al. (1987) subsequently showed that endonucleases D4 and E also recognized urea residues and nicked D N A containing such lesions. For endonuclease E, the g m w a s 3-fold lower for nicking AP DNA compared to urea-containing DNA, although the Vmax values were about the same for both substrates. AP D N A competitively inhibited endonuclease E activity on urea-containing substrates indicating that the nicking activities observed with the AP- and urea-containing substrates were probably mediated by the same enzyme. These results are similar to what this group had previously observed for E. coli exonuclease III (Kow et al., 1985) leading to their speculation that the ability to recognize urea residues may be a general property of certain AP endonucleases. A recently described enzyme, yeast redoxyendonuclease, is similar in many respects to E. coli endonuclease III (Gossett et al., 1988). Experiments utilizing D N A sequencing methodologies have shown that yeast redoxyendonuclease recognizes and cleaves heavily UV-irradiated and oxidized D N A substrates (containing thymine glycol) 3' to the lesion site, producing D N A cleavage products containing 5'-phosphoryl and 3"-modified base free sugar groups (Gossett et al., 1988). On the basis of electrophoretic mobilities of the DNA-scission products in DNA-sequencing gels, yeast redoxyendonuclease also cleaves depurinated D N A in a manner identical to that of E. coli endonuclease III (Doetsch et al., unpublished results). Although yeast redoxyendonuclease (ap-

186 prox. 40 kDa) is somewhat larger in size compared to endonuclease III (approx. 24 kDa), the two enzymes are similar with respect to most reaction parameters (salt and p H effects) and the absence of a divalent cation requirement. Another recently described yeast enzyme, a D N A 3'-repair diesterase, bears a very close resemblance, on a functional basis, to E. coli endonuclease IV (Johnson and Demple, 1988a, b). This enzyme is capable of removing a variety of 3'-esters in D N A including 3'-phosphoglycolaldehyde, 3'-phosphoryl, 3'-a, fl-unsaturated aldehydes as well as D N A polymerase blocking 3'-damages induced by H202 or bleomycin (Johnson and Dempie, 1988b). This yeast 3'-repair diesterase is a protein of M r 40 500, has been purified to homogeneity, and is maximally stimulated by Co 2+ (Johnson and Demple, 1988a). The yeast enzyme also appears to be the major AP endonuclease in yeast cells and hydrolyzes D N A 5' to AP sites to produce 3'-hydroxyl termini that are substrates for D N A polymerase and 5'-deoxyribose 5-phosphate termini. The turnover numbers for the yeast enzyme on the various 3'-damages and AP substrates are within the same range observed with E. coli endonuclease IV (Johnson and Demple, 1988b; Levin et al., 1988). These activities make the yeast D N A 3'-repair diesterase remarkably similar to E. coli endonuclease IV and suggest that, like endonuclease IV, the yeast enzyme m a y participate in two distinct DNA-repair pathways. One pathway would involve the trimming of the 3'-ends of strand break products produced by oxidative and ionizing radiation-induced damage and by the action of certain enzymes that act as fl-elimination catalysts (e.g. endonuclease III). The second pathway would involve the processing of AP sites produced under a variety of conditions. In yeast, the D N A 3'-repair diesterase would function as the major constitutive enzyme involved in the above two steps in D N A repair. In contrast, exonuclease III appears to be the major constitutive enzyme in E. coli involved in the trimming of blocked 3'-termini as well as in the processing of AP sites (Demple et al., 1986). Endonuclease IV can be induced by superoxide generators to levels comparable to those of exonuclease III (Chan and Weiss, 1987) although the yeast enzyme does not appear to be induced by such agents and is a

relatively abundant protein in yeast cells (Johnson and Demple, 1988a). It will be interesting to determine whether or not the close physical and enzymatic similarities shared between the yeast D N A 3'-repair diesterase and E. coli endonuclease IV are reflected in the amino acid sequences of these two enzymes and whether or not similar enzymes exist in other eukaryotes. D. melanogaster A P endonucleases. Two distinct AP endonucleases, I and II that possess different chromatographic properties, have been isolated from D. melanogaster embryos (Spiering and Deutsch, 1981, 1986). The p H optima and other reaction parameters for these enzymes have been determined. AP endonuclease I has a molecular size of 66 kDa, is stimulated by Mg z+, and is inhibited by E D T A whereas AP endonuclease II is slightly smaller (63 kDa) and requires Mg 2+ as a cofactor. Both AP endonucleases I and II cross-react with an antibody prepared against a human H e L a cell AP endonuclease (Kane and Linn, 1981) that is also able to cross-react with E. coli endonuclease IV (Spiering and Deutsch, 1986). D N A substrates containing AP sites that are nicked by AP endonuclease I fire not substrates for D N A polymerase I and there is some evidence to suggest that this enzyme cleaves the phosphodiester backbone 3' to AP sites leaving a 3'-deoxyribose phosphate and a 5'-hydroxyl group that is a substrate for T4 polynucleotide kinase. Such a cleavage mechanism for Drosophila AP endonuclease I is unique for all AP endonucleases characterized to date. In contrast, AP endonuclease II cleaves 3' to AP sites and probably leaves 3'-modified deoxyribose and 5'-phosphoryl termini similar to other AP lyases such as E. coli endonuclease III. Margulies and Wallace (1984) have reported the partial purification and characterization of a Mg2+-stimulated AP endonuclease from D. melanogaster embryos that is sensitive to high salt concentrations and is inhibited by E D T A and N-ethylmaleimide. The relationship of this enzyme to AP endonucleases I and II is not known at this time. Recently, Kelley et al. (1989) reported the cloning of a Drosophila c D N A that encodes a putative AP endonuclease by utilizing a human H e L a cell AP endonuclease antibody (Kane and Linn, 1981)

187 to screen a Drosophila embryonic hgt11 expression library. The c D N A of the Drosophila gene (designated AP3) produced an in vitro translation product of 35 kDa that was identical in size to an AP endonuclease recovered from Drosophila extracts. The C-terminal portion of the predicted protein sequence encoded by AP3 contained presumptive D N A binding domains while the amino terminus showed some similarity to the E. coli recA gene. The 1.2-kb AP3 cDNA mapped to a region on the third chromosome where a number of mutagen-sensitive alleles reside (Boyd et al., 1981). AP3 was expressed as an abundant 1.3-kb m R N A and was detected throughout the life cycle of Drosophila. A second, 3.5-kb m R N A also hybridized to AP3 cDNA, but the presence of this species was restricted to Drosophila early developmental stages. The human HeLa cell AP endonuclease antibody (Kane and Linn, 1981) cross-reacted to proteins of 35, 63 and 91 kDa from partially purified Drosophila AP endonuclease preparations. In addition, Drosophila proteins corresponding to 25, 35 and 63 kDa sizes were extracted from an SDS-polyacrylamide gel and were found to possess AP endonuclease activity. The differences between this finding and an earlier study by this group in which (1)63- and 66-kDa AP endonucleases were isolated and (2) neither the 35- nor the 91-kDa antibody-reactive proteins were detected, were attributed to differences in the protein-purification methods employed in these two studies. The demonstration that the in vitro translated protein possesses AP endonuclease activity will verify the notion that the AP3-encoded gene product encodes an AP endonuclease. Further studies will also be necessary to determine the relationship between the AP3-encoded gene product and the previously identified Drosophila AP endonucleases. A P e n d o n u d e a s e s from mammalian sources

A large number of AP endonucleases have been found in a variety of mammalian sources including rodent, bovine and human cells. In general, these AP endonucleases can be divided into two major groups; (1) those requiring or stimulated by divalent cations that hydrolyze D N A 5' to the AP

site to produce 3'-hydroxyl and 5'-deoxyribose 5-phosphate termini and (2) those active in the presence of EDTA that cleave D N A via a flelimination mechanism (AP lyases) and possess associated D N A glycosylase activities directed against oxidized, ring-modified bases that have lost aromaticity. The enzymes in the first group appear to represent the major mammalian cellular AP endonucleases while the enzymes in the second group are thought to be primarily D N A glycosylases that function in the initial step of the base-excision repair pathway. Within a given species and cell type, there appears to be great heterogeneity amongst the mammalian AP endonucleases and multiple, distinct enzymes have been isolated and characterized. Mouse plasmacytoma A P endonucleases. A number of studies on AP endonucleases have been carried out utilizing the mouse plasmacytoma cell line MPC-11 and have yielded enzymes of the two major types described above. Initial studies showed that there were at least two activities present in MPC-11 cells that cleaved acid-depurinated D N A (Nes and Nissen-Meyer, 1978; Nes, 1980a, b). One of these activities was abolished by E D T A (and simulated by Mg2+), did not act on methylated or OsOa-oxidized D N A and corresponded to a protein of about 28 kDa (Nes, 1980a). The pH and salt optima for this enzyme were found to be 9.5 and 50 m M (KC1), respectively. Recent studies by Haukanes et al. (1988) have examined the mode of phosphodiester bond cleavage mediated by this AP endonuclease. DNA-sequencing techniques were utilized to determine the nature of the 3'and 5'-termini created following cleavage of 3'and 5'-end-labelled, depurinated D N A substrates of defined sequence. This AP endonuclease was found to produce DNA-scission products containing 3'-hydroxyl and 5'-deoxyribose 5-phosphate termini, indicating a cleavage mechanism that involves incision of the C ( 3 ' ) - O - P bond 5' to the AP site. These investigators have extended these studies to include analysis of the base specificity of cleavage of this AP endonuclease on endlabelled, defined sequence single- and doublestranded D N A oligonucleotides of various lengths (Haukanes et al., 1989a). The enzyme acts on AP sites contained in single-stranded oligonucleotides

188 ranging in size from 9 to 40 nucleotides, but not on oligonucleotides 7 nucleotides in length. The maximum rate of AP endonuclease activity on the single-stranded oligonucleotide substrates was about 1 / 3 0 t h that observed for either a doublestranded oligonucleotide of 55 base pairs or ~X174 RF DNA. Analysis of the base specificity of cleavage indicated that AP sites contained within purine-rich regions were preferentially cleaved compared to other regions in doublestranded DNA. Such preferential cleavage was not observed in the single-stranded substrates used. These results suggest that this AP endonuclease has a preference for certain sequences in duplex D N A that may result in more efficient repair of some sites in D N A compared to others. Although the second AP endonuclease activity initially isolated from MPC-11 cells is also a protein of about 28 kDa, it possesses a number of properties different from the MgZ÷-stimulated enzyme (Nes, 1980b). This enzyme is not inhibited by EDTA, indicating the lack of a divalent cation cofactor requirement and it also possesses different p H and salt optima compared to the other MgZ+-stimulated AP endonuclease. In addition, the EDTA-resistant AP endonucleases recognize D N A damaged by high doses of UV light, OsO4, and -/-rays and preferentially cleave supercoiled DNA (Nes, 1980b; Helland et al., 1982, 1985; Kim and Linn, 1989). This enzyme removes thymine glycol from oxidized D N A substrates via an N-glycosylase activity and cleaves the resulting AP site by a fl-elimination mechanism suggesting that it is functionally similar to E. coli endonuclease III and other eukaryotic redoxyendonucleases (Hollstein et al., 1984; Kim and Linn, 1989). This enzyme, which has been designated 'UV endonuclease II' by Kim and Linn (1989) is similar to a second MPC-11 cell enzyme isolated by these investigators and designated 'UV endonuclease I'. UV endonuclease I also possesses thymine glycol D N A glycosylase activity, cleaves AP sites by fl-elimination, nicks heavily UV-irradiated DNA substrates, and is active in the presence of EDTA. However, UV endonuclease I is larger (43 kDa), possesses slightly different optimal reaction parameters for salt, p H and detergent effects, and does not show a preference for supercoiled D N A substrates (Kim and Linn, 1989). These

investigators have suggested that the finding of two related enzymes that have overlapping, but non-identical properties may be analogous to the situation present in E. coli where the functionally related, but non-identical endonucleases III and VIII may mediate similar roles (Melamede et al., 1987; Wallace, 1988). A third type of AP endonuclease isolated from MPC-11 cells is found in mitochondria and preferentially cleaves depurinated, supercoiled D N A substrates (Tompkinson et al., 1988). Two variant forms of the enzyme separate during the course of purification and possess slightly different properties. On the basis of reactivity with the antibody to the human HeLa cell AP endonuclease on immunoblots (Kane and Linn, 1981), the mitochondrial AP endonuclease is a monomeric protein of about 65 kDa arising from a possible precursor of 82 kDa. This property distinguishes it from the other MPC-11 cell AP endonucleases described above. The reaction parameters for this enzyme have been determined and it is stimulated by Mg 2÷. The mitochondrial AP endonuclease preferentially cleaves depurinated supercoiled substrates over relaxed substrates and produces DNA-scission products that serve as primers for DNA polymerase I, suggesting a cleavage mechanism 5' to the AP site which generates 3'-hydroxyl termini. This enzyme appears to share many properties with the other, major mammalian AP endonucleases and Tompkinson et al. (1988) have suggested that it may function in the mitochondria in DNA-repair pathways as well as serve to eliminate damaged mitochondrial genomes from the gene pool. Rat-liver A P endonucleases. A number of studies have reported the purification and characterization of AP endonucleases from rat-liver chromatin (Verly and Paquette, 1973; Thibodeau and Verly, 1980; Thibodeau et al., 1980; Verly et al., 1981). The enzyme requires e i t h e r Mg 2+ o r M n 2+ as cofactors, possesses a pH optimum of 8.0 and is inhibited by high ionic strength. Two forms of this enzyme have been isolated and designated the 0.2 M and 0.3 M isozymes, reflecting the potassium phosphate concentrations necessary to elute them from hydroxyapatite columns (Cesar and Verly, 1983). The 0.2 M enzyme is probably a product of

189 proteolytic degradation because its appearance can be greatly inhibited by inclusion of phenylmethylsulfonyl fluoride in the chromatin protein preparations (Bricteux-Gregoire et al., 1983). Hence the major chromatin AP endonuclease in intact cells is probably the 39 kDa, 0.3 M isozyme (Cesar and Verly, 1983). This enzyme possesses all of the properties ascribed to the chromatin-associated rat-liver AP endonucleases in earlier studies. It does not recognize undamaged or alkylated D N A substrates and it cleaves 5' to AP sites to produce 3'-hydroxyl and 5'-deoxyribose 5-phosphate termini (Cesar and Verly, 1983). The DNA incision mechanism of the rat-liver AP endonuclease has been further studied by Bailly and Verly (1988b) in the context of its role and the roles of r-elimination and 8-elimination reactions in the excision repair of AP sites in mammalian DNA. AP sites that have been nicked via a polyamine- or histone-catalyzed r-elimination reaction result in the production of a 3'-a, fl-unsaturated aldehyde, 4-hydroxy-2-pentenal. The rat-liver AP endonuclease is incapable of recognizing this substrate and thus would be ineffective in the repair of AP sites if the initial cleavage step is a r-elimination reaction. A subsequent 8-elimination reaction however, releases the a, fl-unsaturated aldehyde leaving a 3'-phosphoryl group which can be removed by a chromatin 3'-phosphatase and the resulting single nucleotide gap could be repaired by the combined action of D N A polymerase and ligase (Bailly and Verly, 1988b). These investigators also propose an alternative scenario in which the rat-liver AP endonuclease mediates the initial reaction and cleaves 5' to the AP site leaving 3'-hydroxyl and 5'-deoxyribose 5-phosphate termini. The resulting baseless 5'-sugar can be released by r-elimination (catalyzed by an AP lyase) and repair can be completed by D N A polymerase and ligase as described above. The latter hypothesis is probably the most reasonable since it involves the participation of the major cellular AP endonuclease. It remains to be determined however, whether or not the other major mammalian cellular AP endonucleases are also incapable of recognizing the r-elimination products generated by the AP lyase activities of polyamines and histones. A rat-brain AP endonuclease has been isolated

from neocortex chromatin (Ivanov, 1987; Ivanov et al., 1988). Although this enzyme is smaller (28 kDa) than the major rat-liver AP endonuclease, it requires either Mg 2+ or Mn 2÷ as a cofactor and shares similar reaction parameters with the liver enzyme. The DNA-cleavage products of the ratbrain AP endonuclease are efficient primers for D N A synthesis by D N A polymerase fl suggesting that this enzyme cleaves 5' to the AP site producing 3'-hydroxyl and presumably, 5'-deoxyribose 5-phosphate termini (Ivanov et al., 1988). It was also reported that in vitro, the rat-brain AP endonuclease, together with DNAase B I I I (a rat-brain exodeoxyribonuclease) could excise AP sites in PM2 phage DNA. The resulting gap could be filled with rat liver D N A polymerase fl and E. coil D N A ligase, completing repair. Since both D N A polymerase fl and D N A ligase II have been identified in rat-brain tissue, these investigators proposed that complete repair of AP sites can take place in rat-brain cells. The identification of AP endonucleases and other DNA-repair enzymes in brain tissue is an important first step in elucidating the systems present for maintaining the genetic stability and biological integrity of non-dividing, terminally differentiated tissues. Bovine A P endonucleases. AP endonucleases have been isolated from both calf thymus and liver tissue, with the Mg2+-dependent thymus enzyme being one of the most extensively characterized mammalian AP endonucleases to date. The isolation of a Mg/+-stimulated AP endonuclease from calf thymus was first reported by Ljungquist and Lindahl (1974a, b) and has been subsequently studied by several groups of investigators (Ljungquist et al., 1975; Henner et al., 1987; Sanderson et al., 1988). The enzyme has a broad pH optimum, is optimally active at relatively low salt concentrations, is inhibited by adenine, and is the same size (37 kDa) as the major human AP endonucleases from HeLa cells (Kane and Linn, 1971; Kuhnlein, 1985) and placenta (Shaper et al., 1982). A broadly similar enzyme has also been isolated from calf liver although, unlike the thymus enzyme, it apparently acts on reduced AP sites (Kuebler and Goldthwait, 1977). Comparison of the other properties of the thymus enzyme with the major human cellular AP

190 endonucleases indicates these enzymes are functionally highly conserved between bovine and human cells. Henner et al. (1987) have purified the thymus AP endonuclease to apparent homogeneity and have reported the N-terminal amino acid sequence. The sequence obtained (22 amino-terminal residues) does not, to date, show extensive similarity to the sequences of any other known proteins. The substrate specificity and mechanism of action of the thymus AP endonuclease have been recently investigated in some detail (Sanderson et al., 1988). Utilizing a synthetic duplex D N A oligonucleotide of 22 base pairs containing a single, centrally located abasic site as a substrate, the cleavage products generated by the thymus enzyme have been analyzed using D N A sequencing methodologies. The thymus AP endonuclease incises D N A 5' to the abasic site, producing fragments containing 3'-hydroxyl termini that are efficient primers for terminal deoxynucleotidyl transferase. The 5'-termini presumably contain deoxyribose 5-phosphate moieties. A series of synthetic and natural D N A and oligonucleotide substrates were utilized to determine the structural requirements for the enzyme (Sanderson et al., 1988). These substrates included deoxyribose analogs that were incapable of ring opening or ring closure and substrates that lacked ring structures altogether but retained the phosphodiester backbone. Synthetic AP sites containing ethylene glycol, propanediol, or tetrahydrofuran interphosphate linkages were readily cleaved by the thymus AP endonuclease whereas reduced AP sites, methoxyamine-reacted AP sites, urea and thymine glycol residues were not cleaved by the enzyme. The thymus AP endonuclease was also found to have an absolute requirement for double-stranded D N A , a property that contrasts to the ability of the mouse plasmacytoma major AP endonuclease to cleave abasic sites in single-stranded oligonucleotides (Haukanes et al., 1989a). These results suggest that the bovine-thymus AP endonuclease has no absolute requirement for either ring-opened or ring-closed deoxyribose moieties for its recognition of DNA-cleavage substrates. Sanderson et al. (1988) proposed that the enzyme may interact with the pocket in duplex D N A that results from base loss or with the altered conformation of the phosphate backbone resulting from an AP site.

Hence, there does not appear to be any absolute requirement for a deoxyribose in either recognition or cleavage of a base-free site by this calfthymus AP endonuclease. A calf-thymus enzyme analogous to E. coli endonuclease III has been isolated and characterized (Bachetti and Benne, 1975; Breimer, 1983; Doetsch et al., 1986). The initial studies on this enzyme identified it as an endonuclease capable of cleaving heavily UV-irradiated or X-irradiated D N A substrates in the presence of E D T A indicating no requirement for divalent cations (Bachetti and Benne, 1975). This enzyme is relatively small in size (25 kDa) and possesses a u r e a - D N A glycosylase activity (Breimer et al., 1983). Subsequent studies (Doetsch et al., 1986; Helland et al., 1987) utilizing DNA-sequencing techniques have shown that this enzyme is functionally similar to endonuclease III, acts on both supercoiled and relaxed D N A substrates, and is a thymine g l y c o l - D N A g l y c o s y l a s e / A P endonuclease. Hence, this enzyme is a m e m b e r of the redoxyendonuclease group of D N A - r e p a i r enzymes. The mechanism of D N A cleavage mediated by the calf-thymus redoxyendonuclease appears to be somewhat different, however, from that of E. coli endonuclease III. Calf-thymus redoxyendonuclease cleaves AP sites to produce D N A scission products containing 3'- and 5'phosphoryl termini (Doetsch et al., 1986). This finding suggests that the c a l f - t h y m u s redoxyendonuclease may be an AP lyase cleaving abasic sites via a combined fl,&elimination reaction (Bailly and Verly, 1988b; Bailly et al., 1989a). Further studies on the mechanism of D N A cleavage are necessary to confirm this notion. Human A P endonucleases. H u m a n AP endonucleases have been the most extensively studied group of eukaryotic AP endonucleases. A large number of these enzymes have been identified and characterized from a variety of h u m a n cells and tissues including lymphoblasts, fibroblasts, H e L a cells, placenta and liver. As is the case with the other mammalian AP endonucleases, the h u m a n enzymes isolated and characterized to date are, in general, either Mg2+-dependent and hydrolyze D N A 5' to AP sites to yield 3'-hydroxyl termini, or are Mg2+-independent and cleave D N A 3' to

191 AP sites to yield 5'-phosphoryl termini. Multiple, chromatographically distinct species of AP endonucleases possessing similar activities have been observed within a single cell or tissue type (e.g. placenta) and may represent products of proteolysis generated during purification or, at least in some cases, may represent isozymes with different posttranslational modifications and subcellular targets. Two enzymes from placenta (Shaper et al., 1982; Haukanes et al., 1989b) and one from HeLa cells (Kane and Linn, 1981) have been purified to apparent homogeneity and much is known conceming their physical and catalytic properties. Initial studies with lymphoblasts (Brent, 1975, 1976), HeLa cells (Teebor and Duker, 1975; Duker and Teebor, 1976) and liver tissue (Springate and Liu, 1980) indicated the presence of Mg2+-dependent activities that incised depurinated DNA substrates. The liver enzymes, which have not been extensively studied, exist as 3 chromatographically distinct species ranging in size from 24 to 31 kDa. The liver AP endonucleases have similar reaction parameters, somewhat different KmS for AP sites, and cleave DNA 5' to AP sites to yield 3'-hydroxyl termini (Springate and Liu, 1980). The reaction parameters of the lymphoblast AP endonuclease have been determined and its size (35 kDa) is similar to those of the other major mammalian cellular AP endonucleases. The HeLa cell enzyme is slightly larger, 37.6-41 kDa (Kane and Linn, 1981; Kuhnlein, 1985) and has been purified to apparent homogeneity (Kane and Linn, 1981). The reaction parameters of the HeLa enzyme are similar to those of the other mammalian major AP endonucleases and the enzyme is inhibited by adenine, hypoxanthine, adenosine, AMP, AMPribose and NAD ÷. This enzyme appears to be the major AP endonuclease in HeLa cells, is active on both single and double stranded DNA, and cleaves DNA 5' to AP sites to yield 3'-hydroxyl termini and 5'-deoxyribose 5-phosphate termini. HeLa AP endonuclease is also capable of removing deoxyribose 5-phosphate from AP DNA that contains deoxyfibose at the 3'-terminus (Kane and Lima, 1981). A polyclonal antibody (Kane and Linn, 1981) has been raised against this enzyme and has been extremely useful in the identification of AP endonucleases from other human cell types as well as other species, including Drosophila

(Spiering and Deutsch, 1986; Kelly et al., 1989). 6 chromatographically distinct forms of placental AP endonuclease activities ranging in sizes from 27 to 31 kDa have been reported (Linsley et al., 1977). These enzymes were shown to be stimulated by Mg 2÷ and were catalytically similar to each other with some differences observed for their K m values for AP sites. These activities incised DNA 5' to AP sites to produce 3"-hydroxyl termini. It is likely that the 6 forms of AP endonucleases observed in this study were at least, in part, proteolysis products generated during the course of enzyme purification. A subsequent study by Shaper et al. (1982) under conditions minimizing proteolysis, results in the purification to apparent homogeneity of a single, 37-kDa, Mg 2÷dependent placental AP endonuclease. The mode of phosphodiester bond cleavage mediated by the homogeneous placental AP endonuclease was found to be quite unusual in that the enzyme could cleave DNA either 3' (40% of incisions) or 5' (60% of incisions) to AP sites, producing termini corresponding to those produced by both major types of mammahan AP endonucleases (Grafstrom et al., 1982). This feature is unique amongst all AP endonucleases characterized to date. Recently, the purification and characterization of a second, distinct, Mg2+-dependent placental AP endonuclease has been reported (Wittwer et al., 1989; Haukanes et al., 1989b). This enzyme differs from the 37-kDa placental AP endonuclease in that it is smaller (26.5 kDa) and cleaves DNA exclusively 3' to AP sites to produce 5'-phosphoryl and 3'-deoxyribose (or modified deoxyribose) termini, possibly acting as a t-elimination catalyst or 'class I' AP endonuclease (Haukanes et al., 1989b). Both this enzyme and E. coli endonuclease III generated identical, end-labelled DNA-cleavage fragments when such products were analyzed on DNA-sequencing gels (Haukanes et al., 1989b). The Mg2+-depen dency of this enzyme is unusual since the vast majority of other previously characterized 'class I' AP endonucleases are divalent cation-independent. Further investigations of the 26.5-kDa placental AP endonuclease will be necessary to determine whether or not it possesses a substrate specificity range similar to endonuclease III and to determine its relationship to the previously characterized 37-kDa placental enzyme.

192 Two distinct, Mg2+-stimulated AP endonucleases, designated 'AP endonuclease I' and 'AP endonuclease II' have been isolated from cultured human fibroblasts (Kuhnlein et al., 1976, 1978; Mosbaugh and Linn, 1980). The K m for AP sitecontaining D N A substrates is lower for AP endonuclease I compared to AP endonuclease II. AP endonuclease I cleaves DNA 3' to AP sites to produce termini analogous to those produced by T4 endonuclease V (or E. coli endonuclease III) acting on UV-damaged D N A (Mosbaugh and Linn, 1980), suggesting that this enzyme is a 'class I' AP endonuclease. The mode of phosphodiester bond cleavage for AP endonuclease I and its stimulation by Mg z+ are analogous to the properties of the recently characterized 26.5-kDa placental AP endonuclease described above (Haukanes et al., 1989). In future studies, it will be interesting to determine what relationships exist between these two enzymes. AP endonuclease II, on the other hand, appears to cleave DNA 5' to AP sites and produce termini (3'-hydroxyl and 5'-deoxyribose 5-phosphate) similar to the other major, Mg2+-de pendent mammalian AP endonucleases. AP endonuclease I also appears to be absent in cultured fibroblasts from patients with xeroderma pigmentosum complementation group D (Kuhnlein et al., 1979; Mosbaugh and Linn, 1980). A large number of reports have indicated that an enzyme similar to E. coli endonuclease III is present in various types of human cells including fibroblasts, lymphoblasts, and HeLa cells (see Table V, Wallace, 1988 and refs. cited therein). Initial studies with lymphoblasts (Brent, 1983) and HeLa cells (Teebor and Duker, 1975; Duker and Teebor, 1976) indicated the presence of a Mg 2÷independent enzyme that cleaved DNA damaged by high doses of UV light, y-rays, OsO 4, or acid. This enzyme, human redoxyendonuclease, has been characterized in some detail from lymphoblasts (Brent, 1983; Doetsch et al., 1987; Lee et al., 1987). With the exception of possessing a larger size (60 k D a ) , h u m a n l y m p h o b l a s t redoxyendonuclease is similar to the other mammalian redoxyendonucleases discussed in previous sections. The enzyme has no divalent cation requirements and is a thymine glycol-DNA glycosylase/AP endonuclease (Lee et al., 1987). Brent (1983) has shown that this enzyme incises D N A to

produce fragments that are poor primers for D N A polymerase I. Lee et al. (1987) have employed DNA-sequencing techniques for the analysis of the enzyme-generated DNA-cleavage products and have shown that the enzyme incises AP D N A substrates in a manner identical to that of the calf-thymus redoxyendonuclease (Doetsch et al., 1986). The human redoxyendonuclease-generated DNA-strand scission products contain 3'- and 5'phosphoryl termini products possibly by a combined /3,8-elimination reaction, making this enzyme a putative AP lyase (Bailly and Verly, 1988b; Bailly et al., 1989a). Additional mechanistic studies are necessary to confirm that enzymes such as the human redoxyendonuclease and its functional analogs from other mammalian species are, in fact, AP lyases. In conclusion, a substantial amount of information exists on the physical and catalytic properties of many of the eukaryotic AP endonucleases identified to date. An understanding of the relationships of these enzymes isolated from numerous species ranging from yeast to humans will be greatly facilitated by the cloning, isolation, and characterization of the corresponding genes and determining their regulation under a variety of cell growth conditions. Such studies are currently beginning or are already underway in a number of laboratories.

Possible mechanisms for phosphodiester bond cleavage by AP endonucleases and AP lyases A few simple generalizations can be made after examining the well-characterized AP endonucleases and AP lyases from both prokaryotes and eukaryotes. The AP endonucleases, which cleave 5' to AP sites, require a metal ion for activity, usually Mg 2+, Ca 2+ or Mn 2+. The AP lyases, which cleave 3' to AP sites and which have associated N-glycosylase activities, do not require metal ions for activity and are EDTA-resistant. The cleavage catalyzed by AP endonucleases is hydrolytic while the cleavage catalyzed by AP lyases proceeds by a r-elimination reaction. Studies with other nucleases and with tripeptides suggest possible reaction mechanisms for AP endonucleases and AP lyases.

193

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Staphylococcal nuclease and bovine pancreatic deoxyribonuclease I (DNAase I) have been extensively studied. The structures of the enzymes have been determined (Cotton et al., 1979; Suck and Oefner, 1986) and the stereochemical courses of the reactions catalyzed by these enzymes have been determined (Mehdi and Gerlt, 1982; Mehdi and Gerlt, 1984). DNAase I is a Ca2+-dependent endonuclease which acts on single- and doublestranded D N A to yield a nick bordered by 5'phosphoryl and 3'-hydroxyl termini. Suck and Oefner (1986) have proposed a mechanism of action for DNAase I in which a carboxylate anion accepts a proton from a histidine which accepts a proton from water. The resulting hydroxide ion attacks the phosphorus in the phosphodiester bond ultimately cleaving the P - O - 3 ' bond. The Ca 2+ is assumed to position the enzyme with respect to the phosphodiester bond and to facilitate the nucleophilic attack of the hydroxide ion. Staphylococcal nuclease has Ca2+-dependent exonucleolytic and endonucleolytic activity on both D N A and RNA. The endonucleolytic activity

yields a nick bordered by a 3'-phosphoryl and a 5'-hydroxyl group. Models for the mechanism of action of staphylococcal nuclease (Cotton et al., 1979; Hilber et al., 1987; Serpersu et al., 1987) propose that the carboxylate group of a glutamate residue serves as a general base to abstract a proton from water creating a nucleophilic hydroxide ion. The hydroxide ion attacks the phosphorous leading to cleavage of the P - O - 3 ' bond. The Ca 2+ ion in staphylococcal nuclease is proposed to form an ionic bond with the 5'-phosphate oxygen atom and make the phosphate more susceptible to nucleophilic attack. In both reactions, there is inversion of configuration at the phosphorus (Mehdi and Gerlt, 1982, 1984) suggesting that the reactions proceed by a single displacement. It seems possible that AP endonucleases may use a similar reaction mechanism. Scheme 1 s u m marizes a reaction mechanism for an AP endonuclease based on the mechanisms discussed above. A metal ion forms an ionic bond with a phosphate oxygen atom to make the phosphodies-

194

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195

ter bond more susceptible to attack and to orient the enzyme on the DNA. General base catalysis results in proton abstraction from water with the hydroxide ion attacking the phosphate to form a pentacovalent intermediate. The P - O - 3 ' bond is then broken. The metal ion may also facilitate the leaving of the oxyanion group (Herschlag and Jencks, 1987). The straightforward attack of water on the phosphodiester bond is not the only mechanism by which this reaction can proceed. The nucleophilic attack of a carboxylate group could lead to the formation of an acyl phosphate ester intermediate (Mehdi and Gerlt, 1982). The nucleophilic attack of water on the carboxyl carbon of the intermediate would result in hydrolysis of the phosphodiester bond with inversion of configuration at the phosphorus while the nucleophilic attack of water on the phosphorus in the intermediate would result in hydrolysis with retention of configuration at the phosphorus (Gerlt et al., 1983). Both stereochemical analysis and high resolution X-ray-structure analysis will be required to determine the exact reaction mechanisms used by AP endonucleases. As described in a previous section of this review, model studies show that a fl-elimination reaction can occur at AP sites. This type of reaction can proceed by a proton abstraction or by a Schiff base intermediate. There is evidence that the Schiff base intermediate is formed in a simple model system (Bertrand et al., 1989) and Kow and Wallace (1987) have proposed a mechanism of action for endonuclease III which has a Schiff base intermediate. Since carbonyl groups are activated by iminium ion (Schiff base) formation, we will focus our discussion of mechanisms on those in which an iminium ion is formed. The most common reaction is initiated by addition of a lysine amino group to a carbonyl group on the substrate (Hupe, 1984) as shown in Scheme 2. The addition requires the attack of an unprotonated amine on the carbonyl to form a charged intermediate which accepts a proton to form a neutral carbinolamine. A dehydration reaction results in the formation of the iminium ion product. The protonated imine is susceptible to a nucleophilic attack. A base-catalyzed proton abstraction at the a-carbon occurs readily to initiate a fl-elimination

(Page, 1984). A hydrolytic reaction then releases the amino group of the lysine and yields the 4-hydroxy-2-pentenal at the 3'-terminus. The active site for an AP lyase may be expected to have one or possibly two lysine residues since the base catalyzing the proton abstraction can be another amine. Alternatively, a hydroxide ion or the 3'phosphodiester leaving group itself (Widlandski et al., 1989) could act as the base. High resolution X-ray analysis and further mechanistic studies will ultimately define the mechanism of action of AP lyases. Other possible mechanisms of action include the transiminization mechanism proposed by Kow and Wallace (1987) for endonuclease III and a modified version of the mechanism outlined above that would allow for a fl,8-elimination.

Acknowledgments We thank John Gerlt for helpful discussions concerning the possible reaction mechanisms for AP lyases and D N A N-glycosylases. Our research on AP endonucleases is supported by N I H grants GM33346 (R.P.C.) and CA42607 (P.W.D). P.W.D. is a recipient of a Research Career Development Award (CA01441) from the National Cancer Institute.

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