Chemical nucleases: new reagents in molecular biology.

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Annu. Rev. Biochem. 1990.59:207-236. Downloaded from www.annualreviews.org Access provided by New York University - Bobst Library on 02/02/15. For personal use only.

CHElVUCAL NUCLEASES: NEW REAGENTS IN MOLECULAR BIOLC)Gy1 David S. Sigman

2

and Chi-hong B. Chen

Department of Biological Chemistry, School of Medicine, Department of Chemistry and Biochemistry, Molecular Biology Institute, University of California, Los Angeles, California 90024 KEY WORDS;

chemical

nucleases,

DNA

SCISSIOn,

site-specific

nudeases,

1,10-

phenanthroline-copper, nucleic acid conformation.

CONTENTS INTRODUCTION.....................................................................................

208

Synthetic Nucleolytic Agents.................................................................... B iological Nucleolytic Agents .. . . . .......... .... . . . . . ...... ... ...... . . .. . . .... .. . .. . .. .. ... . ... Kinetic Scheme of Chemical Nucleases. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . .

208

PROBES OF CONFORMATIONAL VARIABILITY OF NUCLEIC ACIDS ............ DNA...... . ... ... ....... ... .............. .... . ....... .... .. ... ........ . .. ... ... ... ...... . . . . ... ... .. . 1. 1,10-phenanthroline-copper ...... ... .. .... ... . ...... . ...... ... ... ..... . ..... ...... .. .. ... 2. Octahedrall,JO·phenanthroline-metal complexes . . ... ... ................ .. ...... ... 3. FerrOlls-EDTA as aprobefor conformational variability.......................... RNA. . .... ... . ....... ... .. ...... ...... . .. . .... ... . ....... . . . .... ...... ... ......... . . ...... ... .. . ... . . 1. 1,10-phenanthroline-copper and methidium-propyl-EDTA.. ..... . .. ...... . .... .... 2. Ferrous-EDTA....... . .... .. ... ... .... . ....... .... ....... ...... ... ... ...... ..... ... ... ... ...

211 211

CHEMICAL NUCLEASES AS FOOTPRINTING REAGENTS OF DNA AND RNA.

Comparison to DNase I......................................................................... I. Advantages relative to DNase I .. ... ............. .. " . . ". . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Light-activated uranyl acetate ...... . . ....... .. .. ... " . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. F ootprinting DNA -protein complexes following gel retardation llsing OP·Cu and MPE . ................................................... RNA-Protein Complexes Studied with Uranyl Acetate and OP-Cu.. ...... ... . . . . . . . . ..

208 209

211 213

215

216 216

216 218 218 218 220 220

221

I Abbreviations used: OP-Cu, 1, lO-phenanthroline-copper; MPE, methidi um-propyl-EDTA 2Author to whom correspondence should be addressed.

207

0066-4154/90/0701-0207$02.00

208

SIGMAN & CHEN

SITE-SPECIFIC NUCLEASES.....................................................................

DNA Binding Drugs as Targeting Reagents................................................. Polynucleotides as Targeting Ligands........................................................ 1. Watson-Crick hybridization.................. ........ . .................................... 2. a-Deoxyoligonucleotides.. . ........ . .... . ........ .. . ...... ......... ............... ........ 3. Triple-helixformation.............. . . ... . . . . . . ..... . .. . . . ..... . . . . .. . . . .. . . .. . . . .. . . . . 4. R-Ioops . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . DNA-B indingProteins as Affinity Ligands.......... ......................................... Chemically Synthesized Peptidesfor Directing Scission..................................


CONCLUSIONS/FUTURE DIRECTIONS.......................................................

222 222 223 223 225

22S

229 229 233

234

INTRODUCTION In biological systems, ribonucleases and deoxyribonucleases accomplish the scission of the phosphodicstcr backbone of RNA and DNA by catalyzing nucleophilic attack on the scissile bond via an in-line SN-2 mechanism. In the past 10 years, a group of redox-active coordination complexes have been discovered that cleave the phosphodiester backbone of nucleic acids at physi ological pHs and temperatures by oxidation of the deoxyribose or ribose moiety (l, 2).

Synthetic Nucleolytic Agents Coordination complexes that accomplish strand scission in the presence of oxygen or reducing agents (Figure 1) include the tetrahedral 1,10phenanthroline-cuprous ion, fcrrous-EDTA either free (3) or linked to DNA binding ligands (2) , and metalloporphyrin derivatives (4, 5 ) . DNA scission by uranyl acetate (6) and by the octahedral complexes of 1, lO-phenanthroline and its derivatives prepared from ruthenium or cobalt is light dependent (7). Biological Nucleolytic Agents Two natural products isolated from strains of streptomyces, bleomycin and neocarzinostatin, achieve phosphocliester backbone scission by nonhydrolytic mechanisms. Bleomycin, with iron as a cofactor, cleaves DNA in an oxygen dependent reaction in which the primary site of attack is the C-4 hydrogen (8). Neocarzinostatin, which is composed of a protein and a diacetylene containing cofactor, cleaves DNA at T and A residues at the C-5 of the deoxyribose via hydrogen atom abstraction yielding a 5-aldehyde as an intermediate (9). Calicheamycin y/ isolated from fermentations of Micro monospora echinospora ssp calichensis reacts similarly to neocarzinostatin without a protein cofactor ( 1 0) (Figure 2). This review focuses on the applications and potential of the synthetic and nucleolytic agents in molecular biology. They will be designated "chem ical nucleases," even though in most cases the reagents have been used in excess relative to the target nucleic acid and catalysis has not been

209

CHEMICAL NUCLEASES

b Annu. Rev. Biochem. 1990.59:207-236. Downloaded from www.annualreviews.org Access provided by New York University - Bobst Library on 02/02/15. For personal use only.

o

H �N H

to

0

.... ...- N,I�0 \ �N--Ft N I I "!r--- 0

c

e

Figure 1

o

\

__

°

d

f Structures of synthetic chemical nucleases. (a) 2: 1 1,1 O-phenanthroline-cuprous ion

(Op-eu); (b) methidium propyl-EDTA-iron II (MPE); (c) ferrous-EDTA;

(d) tris (4,7-diphenyl

Tris(l, IO-phenanthroline)ruthenium (II); (e) meta1\oporphyrins; if) uranyl acetate.

rigorously demonstrated. Chemical nucleases have three general uses. First, they are probes of conformational variability of DNA and RNA. Second, they serve as footprinting reagents and can complement the widely used DNase I in this application. Finally, they have permitted the design of site-specific nucleolytic agents in which the chemical nuclease activity is tethered to a carrier ligand with affinity for a particular aspect of nucleic acid structure.

Kinetic Scheme of Chemical Nucleases The kinetic scheme for the scission chemistry by a chemical nuclease (R) involves reversible complex formation followed by the nucleolytic reaction.

210

SIGMAN & CHEN kJ

}(d

R + DNA

�

R-DNA � nicked products

1.

At nonsaturating concentrations scission

of R, the second-order rate constant for (k2) at any sequence position is kl/}(d. Unlike true enzymes, in which

binding and catalysis are integrally interrelated, binding directs the scission


chemistry but does not activate it. The redox chemistry of the chemical nuclease r esponsib le for scission proceeds in solution whether the reagent is

bound to DNA or not. If kJ is constant, then Kd governs the second-order rate constant for scission at any sequence position and binding is the primary determinant of specificity.

The scission of nucleic acids by chemical nucleases shares important similarities with photoaffinity labeling of enzyme active sites

Figure 2

(11). Specificity

Stmcture of naturally occurring chemical nucleases. (a) Bleomycin; (b) Cofactor of

neocarzinostatin; (c) Calicheamycin.


CHEMICAL NUCLEASES

211

in both cases arises from the binding of a carrier, but the chemistry is mediated through highly reactive intermedites. Selectivity is lost if the reac tive intermediates activated in solution are not quenched. Very reactive nonselective intermediates generated in solution react with buffer components and never reach the targets. Less reactive species may require protective quenchers to block their diffusion to the target. Ferrous-EDTA has been widely used as a chemical nuclease activity (12). Unlike op-eu and the more closely related MPE, this reagent, because of its net negativ{: charge, has been proposed to act in solution as a generator of diffusible hydroxyl radicals. It would provide an exception to the kinetic scheme summarized in Equation I. Recent studies have claimed, however, that ferrous·-EDTA cuts single-stranded DNAs less efficiently than double stranded DNAs (13). Possibly even in this case, DNA scission proceeds through a weak complex (high Kd) formed with ferrous-EDTA or an in termediate iron-oxo complex.

PROBES OF CONFORMATIONAL VARIABILITY OF NUCLEIC ACIDS DNA Chemical nucleases that have provided insight into the conformational vari ability of DNA include 1, IO-phenanthroline-copper, the delta tris 4,7diphenyl-I,IO-phenanthroline-cobalt (III) complex, and ferrous-EDTA. I, IO-PHENANTHROLINE-COPPER The 1,1O-phenanthroline-copper complex (OP-Cu) was the first chemical nuclease discovered that cleaved DNA in a reaction that was funnelled through an essential noncovalent intermediate (I). The tetrahedral cuprous complex binds in the minor groove of DNA where it is oxidized by hydrogen peroxide to form a copper-oxo species that may be directly responsible for oxidation of the deoxyribose (14) (Figure 3). The chemistry of scission has been extensively studied, and the reaction pathway a summarized in Figure 4 accounts for 70-90% of the reaction depending on the primary sequence. Stable products in this scheme that have been identified include the free base, 3' - and 5' -phosphoryl ends, and 5methylenefuranone (5-MF) (15-17). Since this reaction mechanism involves initial attack on the C-l H of the deoxyribose that is recessed in the floor of the minor groove, the cvordination complex must bind in this structural domain. The generation of variable but measurable amounts of 3' -phospho glycolates (pathway b) is also consistent with this binding site of the coordina tion complex. Although the formation of base propenals is stoichiometric with phosphoglycolates in the scission of DNA by bleomycin, base propenals have not been detected in OP-Cu scission (17).

212

SIGMAN

&

�::�: ::� :: / r {OP)2CU+


(OP

CHEN

+

DNA .

-

D

O

u ,

{OP)2Cu+--DNA PhC

T

"DNA

� {OPhCu+++=O--DNA

+

OH"

+

H+

OP Figure 3

Kinetic mechanism of the chemical nuclease activity of I, IO-phenanthroline-copper

(OP-Cu).

The efficiency of OP-Cu is dependent on the secondary structure of DNA ( 18). Single-stranded DNA is not an efficient substrate. B-DNA is preferred relative to A-DNA, and Z-DNA (e. g . poly dG-C) is not detectably cut under conditions where B-DNA is readily cleaved ( 19) . This secondary structure indicates that the minor groove of B-DNA provides a preferred binding site 'for the tetrahedral coordination complex. Since the chemistry of the cleavage involves attack on the deoxyribose, the OP-Cu reaction is not specific for the nucleotide at the site of scission. Nevertheless, the scission pattern of any restriction fragment exhibits se quence-dependent reactivity (20-26). The structural features of the minor groove that govern the sequence specificity of this cutting reaction have not been identified even though oligonucleotides of known 3-D structure have been studied. Statistical approaches have revealed that 70% of the variability in the scission rates can be attributed to the nucleotide 5' to the cutting site, although the nucleotide 3' has an impact as well (27). For example, TAT is cleaved more efficiently than TAC at the central A residue; the tetranucleotide TGGT is cleaved more efficiently than TGGC (28, 29). The structural variation of the minor groove reflected in the sequence dependent scission can be associated with biological function. The wild-type lac promoter, which requires the upstream binding of the cyclic AMP binding protein for transcription, has the sequence on the template strand -8 to - 13 of 5'-ACATAC-3' and exhibits two strong cut sites at positions - 13 and -12. The sequence of the strong cyclic AMP-independent UV -5 promoter

CHEMICAL NUCLEASES

O-p-0 O }

0

\

CH2

[0]

B

0

C-l

H U 0

H Annu. Rev. Biochem. 1990.59:207-236. Downloaded from www.annualreviews.org Access provided by New York University - Bobst Library on 02/02/15. For personal use only.

pathway b � �

0

C-41

H

\ CHz

I

I

+

o· p-IQ)

0 C H2 ,

+

unknown corbon frogment

\

�

S'end

1� 0

Q

0

H -

II

o-p-o

H

0

=o

S'end o II

o-P-o ,

o

�

H

,

�

�

o-p=o ,

0 '

3 end Figure 4

B

I

� �

+B

+

"

o-p::o

a

[0]

o�c'o 3

..

\

0

o-p-o ,

,

pathway

,

O-P-O

O-p-0 O CH2 0 O M = (0 H � �

II

,

213

H H-C� C \

i'-

0

H+

=0

H H 5-MF

Reaction scheme for the scission of DNA by op·eu (15).

from - 8 to - 1 3 on the template strand is 5 ' -TTATAC-3' but has four strong scission sites from positions -13 to -10 (Figure 5). The altered minor groove geometry, reflected in increased susceptibility to OP-Cu scission, is also associated with stronger RNA polymerase binding (23, 24). 1,IO-PHENANTHROLINE-METAL COMPLEXES Barton and colleagues have shown that octahedral complexes prepared from 1,10phenanthroline derivatives can bind to DNA (7, 30--3 2). Although octahedral complexes do not exhibit thiol-dependent scission, the rhodium, ruthenium, and cobalt complexes can direct scission adjacent to their binding sites by serving as photosensitizers for the formation of singlet oxygen. Intriguing specificity has been achieved with the enantiomers of octahedral complexes formed by three 1 , lO-phenanthroline derivatives and one metal ion (Figure 6). For example, the delta isomer of the ruthenium complex prepared from 4,7 -diphenyl- l , lO-phenanthroline (DIP) binds preferentially to B-DNA when hypochromism in the charge transfer band is used as an assay (33). In

OCTAHEDRAL

214

SIGMAN & CHEN


13121110

UV-5

A G Gee G AG CAT A C A A C Ae Ace T T A A C I

I

TT -

20

15

10

-WT

-UV-5 5

-1 +1

5

Template Strand Figure 5 Comparison of O P-Cu scission patterns ofUV-5 and wt lac operators . The sequence of wt DNA is indicated . The nucleotide changes of the UV-S mutation are shown [adapted from (24)].

contrast, both enantiomers bind equivalently to poly dG-dC in the Z-fonn using the same method of assay. This observation suggests that the lambda isomer binds preferentially to Z-DNA. The important difference between octahedral and tetrahedral complexes of 1, l O-phenanthroline (copper) is that the fonner bind to the major groove while the latter bind to the minor groove. Sequence-specific binding of the lambda isomer on functional DNA frag ments can be inferred using the light-dependent nicking reaction of the cobalt

CHEMICAL NUCLEASES

2+


2+

Figure 6

215

Enantiomcrs of Tris(l, lO-phcnanthrolinc)ruthenium (II).

complex. Preferential sites of scission arc noted in regions of transcription termination in bacterial plasmids. Although these experiments are insufficient to infer Z-structure in these domains, they do indicate that the sequence dependent variability of DNA can generate high-affinity binding sites for these octahedral complexes FERROUS-EDTA

AS

A

(7).

PROBE

FOR

CONFORMATIONAL

VARIABILITY

Dervan and colleagues introduced ferrous-EDTA for the controlled scission of DNA in their synthesis of MPE (Figure function to

a

I ) (2). By tethering the cleavage

weIl characterized intercalating agent, they ensured that MPE,

like OP-Cu, would form an essential noncovalent intermediate during the course of scission. It nicks double-stranded DNA with little sequence depen dence. Cold carrier DNA is added to block scission of the 32P-target DNA from unbound MPE. Tullius and colleagues

(12) have developed the negatively charged ferrous

EDTA complex as a probe of DNA-ligand binding as well as a reagent to study the conformational variability of DNA. Central to this approach was that the rate of scission at any given position would be a function of the accessibility of diffusible hydroxyl radicals to the deoxyriboses and not the binding of the reagent to DNA. Previous studies on the mechanism of scission of DNA by hydroxyl radicals generated by cobalt60 l' irradiation had demon strated that the primary products were free hases,

5' -phosphorylated ends,

3' -phosphorylated ends, and 3' -phosphoglycolates. The phosphoglycolate

products probably arise from attack of the hydroxyl radical on the C-4 hydrogen atom, while the 3 I -phosphomonoester termini may be derived from attack on the Col hydrogen atom (34). Ferrous-EDT A generates an even cutting pattern in mixed-sequence DNA. However, interesting differences in the scission patterns of oligomers


216

SIGMAN & CHEN

with the sequences 5 ' -CGAAAATTTT-3' and 5' -CGTTTTAAAA-3' have been observed (35, 36) . There is a clear sinusoidal cutting pattern with the former sequence but not the latter. Neighboring mixed-sequence DNA lacks the systematic sinusoidal variation. Differences in the conformation of these two segments are anticipated because double-stranded oligomers of the 5' GAAAATTTTC-3' migrate more slowly than those of 5' -CGTTTTAAAA-3' and have been postulated to adopt a bent conformation (37). The cutting pattern indicates that the minor groove geometry is systematically altered in the bent DNA and that the environment of each adenine is distinct. Arguments that the linear conformation of 5' -CGTTTTAAAA-3' results from the cancellation of successive perturbations involving the AA sequence are not supported by these results. These conclusions should be independent of the detailed mechanism of scission by ferrous-EDTA .

RNA \,lO-PHENANTHROLlNE COPPER AND METHIDIUM-PROPYL-EDTA Computer generated secondary structure maps of RNAs rely on the identification of single- and double-stranded regions using chemical and enzymatic reagents and interspecies primary sequence comparisons (38). OP-Cu, MPE, and ferrous-EDTA provide an additional perspective for evaluating the conforma tion of RNA . In contrast to its reaction preference with DNA, OP-Cu exhibits a strong preference for the single-stranded loops of stem-loop structures in RNA (39) . In general, MPE has a reactivity pattern with RNA that is complementary to OP-Cu, preferring double-stranded regions as would be expected from an intercalating agent (40, 4 1 ) . Neither specificity is absolute, as the sites of scission on tRNAphe indicate (Figure 7). MPE does not react in all double stranded regions. The scission in single-stranded regions by OP-Cu is pre ferred, but not all single-stranded regions are attacked nor are all reactive sites in single-stranded regions. For both reagents, cleavage sites must be adjacent to a binding site . Single-stranded regions generally will provide these binding sites for (OPhCu+, but specific tertiary folds of RNA such as the joint of the L-region (42) may generate strong binding sites as well.

The ferrous-EDTA reaction provides an experimental approach to examine the solvent accessibility of the phosphodiester backbone of RNA, even though more vigorous conditions are required to cleave RNA than DNA (43 ) . The stabilization of the conformation of yeast tRNAphe by magnesium ion can be detected by this reagent. In the absence of magnesium ion , tRNAphe is cut at all sequence positions with equivalent intensities . Upon the addition of magnesium ion, three sequence regions-58-60 (AUe), 48--49 (CC), and 1 8-20 (GGG)-are protected against scission (Figure 7) . FERROUS-EDTA

CHEMICAL NUCLEASES


T arm

64

217

5

57 . 19-..-1·

70 69

D arm

Figure 7 Comparison of the sites of scission of tRNAphe (39, 43). Arrows indicate OP-Cu; circled x's indicate MPE; stars indicate sites protected by magnesium ion from scission by ferrous-EDTA.

Magnesium ions apparently stabilize a conformation in which these residues are in the interior. It is of interest to note that sequence positions 58-60 and 48-49 are in single-stranded regions that are not reactive to OP-Cu. This region must be similarly inaccessible to the larger tetrahedral (OPhCu+ . The scission of a ribozyme derived from Tetrahymena ribosomal RNA intervening sequence by ferrous-EDTA has revealed a variety of sequence


218

SIGMAN & CHEN

positions that are protected from scission by the addition of magnesium ion. Truncated catalytically inactive ribozymes have few sequence regions that are protected by scission even in the presence of added magnesium ion . Mainte nance of a catalytically active conformation with the precise orientation of residues, often remote from one another in primary sequence, is clearly important for all enzymes whether they are RNAs or proteins. Combined use of different reagents should provide an approach for investigating these functionally essential aspects of RNA conformation . CHEMICAL NUCLEASES AS FOOTPRINTING REAGENTS OF DNA AND RNA Comparison to DNase I Chemical nuclcases can serve as nucleolytic agents to identify sequence-specific contacts between ligands and DNA . In this application , they supplement DNase I and base-specific chemi cal modification reagents (e .g. dimethyl sulfate) as "footprinting" agents. D Nase I has proven to be a reliable method to define sequence-specific contacts between proteins and DNA (44) . The chemical nuclease activities of MPE and ferrous-EDTA possess two advantages relative to DNase I as a footprinting reagent. First, since their scission patterns are not highly se quence dependent, they are better suited for defining the limits of recognition sequences for protein binding . For example, if DNase has few cutting sites in the region of a protein's recognition sequence, protection is obviously diffi cult to evaluate . Second, their smaller sizes permit scission closer to the edge of DNA sequence protected by protein binding and therefore a more precise definition of it. Independent of the mechanism of scission of ferrous-EDT A, this reagent has less stringent steric requirements than DNase I , MPE, or OP-Cu. In a valuable review , Tullius has compared the footprints of the lambda repressor with the lambda phage OR-I operator obtained with these four reagents (45) (Figure 8). The higher resolution evident with ferrous-EDTA relative to the other reagents permits the inference that protein binding occurs on a single side of the helix. This information is not evident from the protection patterns obtained with the other footprinting reagents. One novel feature of the activity of OP-Cu as a footprinting reagent not shared by DNase I or other chemical nuclease activities is its detection of a single-stranded template DNA formed by Escherichia coli RNA polymerase at kinetically competent transcription start sites (24, 46). Cutting is not observable on the nontemplate strand. Possibly, this specificity of OP-Cu arises from the binding of the coordination complex to the protein as well as

ADVANTAGES RELATlVE TO DNASE I

2 19

CHEMICAL NUCLEASES

DNase

3

+

4

5

G

MPE· Fe

+

Cu (phen)2 +

-----

6

7

8

9

10


2

+

Fe(EDTA)

Figure 8 Footprints of lambda repressor binding using four nudeolytic agents (45). Lane 1 : control DNA; Lanes 2 and 3 ; DNase I ; Lanes 4 and 5 : ferrous-EDTA; Lane 6 : Maxam-Gilbert G-specific scission; Lanes 7 and 8; MPE; Lanes 9 and 10: OP-Cu. Original photograph kindly provided by T. Tullius.

220

SIGMAN

&

CHEN

fonnation of a pi complex with the single-stranded DNA . The cuprous complex of 5-phenyl- l , lO-phenanthroline is especially efficient at cleaving single-stranded DNA fonned at productive transcription start sites, even though it is slightly less effective than OP-Cu at cleaving B-fonn DNA (T. Thederahn et aI, in preparation) . Although advantages of using fer rous-EDT A as a footprinting reagent have been outlined above, its scission chemistry is quenched by glycerol, which is commonly used in the isolation and storage of DNA-binding proteins. Although this difficulty can be circum vented, an intriguing new reagent, photoactivated uranyl acetate, has been discovered that generates sequence-neutral digestion patterns with random sequence DNA (6) . This light-dependent reagent is not quenched by glycerol , possibly because the linear UO� + cation is a magnesium ion analog that binds to the phosphodiester backbone of the nucleic acid. It provides a footprint of lambda repressor binding to OR-l similar to that obtained with ferrous EDTA .


LIGHT-ACTIVATED URANYL ACETATE

FOOTPRINTING DNA-PROTEIN COMPLEXES FOLLOWING GEL RETARDATION

OP-Cu and MPE also can be used in one type of footprinting experiment for which DNase I is not suitable. Gel-retardation or mobility shift assays provide a powerful method for analyzing DNA-protein interactions (47 , 48) . In this procedure, the binding of a protein to a specific fragment results in the retarded migration of a DNA fragment in a nondenatur ing acrylamide gel. To detennine the specific sequences contacted by the protein, two strategies have been adopted. In one approach, a DNA fragment suspected to contain the binding site of the protein is used as a competitor of the probe DNA for a protein in the preincubation mixture prior to electrophoretic separation. Loss of the retarded band suggests that the com petitor fragment contains the correct sequence [e.g. (49)]. Alternatively , the protein-DNA complex is reacted with DNase I or dimethyl sulfate prior to electrophoresis. The zones of protection evident in the DNA of the retarded complex indicate the binding site The chemical nuclease activities of OP-Cu and MPE provide a simpler and more direct approach to identifying sequence-specific contacts between the protein and DNA. Following the separation of the DNA-protein complexes of interest, the protein-DNA complex is digested with the scission reagent while still embedded in the gel matrix (51 ). Since the coordination complexes are small and readily diffusible , the protein-DNA complex is accessible to the reagent. Footprints are obtained after elution of the oligonucleotide products from the retardation gel and analysis on a sequencing gel. This procedure was originally developed using OP-Cu with the lac repressor-operator interaction USING OP-CU AND MPE

(50).


CHEMICAL NUCLEASES

221

as the test system. Footprints were the same whether the reaction was carried out in solution or within the gel matrix. The advantages of this in-gel footprinting procedure are threefold. First, protein-DNA complexes of defined stoichiometry are examined. In solution footprinting, it is always possible that the protection actually represents a composite of more than one complex. Second, direct footprinting has the advantage relative to oligonucleotide competition in the analysis of mUltiple retarded bands frequently isolated in gel retardation experiments in complex systems. For example, multiple bands can arise from the binding of transcrip tion factor (e.g. EI-A) to a protein that makes sequence-specific contacts with DNA, or from two proteins binding to distinct DNA sequences in a coopera tive manner [e.g. (52-57»). Although both complexes would be lost by competition with oligonucleotides, these possibilities can be readily dis tinguished by footprinting within the gel. Finally, functional protein-DNA complexes are separated in the gel retardation assay and can be studied further. For example, the RNA polymerase-induced changes in the DNA structure can be detected by OP-Cu in the gel matrix (51).

RNA-Protein Complexes Studied with Uranyl Acetate and OP-Cu Most reagents available for footprinting RNA-protein interactions are very specific in their scission of RNA. For example, T l ribonuclease preferentially hydrolyzes single-stranded G residues (58); a-sarcin hydrolyzes purine re sidues in both double- and single-stranded regions (59). Since oxidative chemical nucleases attack the ribose moiety and cleave at all nucleotides, they are potentially useful as RNA footprinting reagents. Light-activated uranyl acetate has recently been used to study the binding of proteins to RNA (60). Light-activated uranyl acetate reacts with RNA in a sequence- and secondary structure-independent reaction. Even in the pres ence of magnesium ion (in contrast to ferrous-EDTA), equivalent cutting is observed at all sequence positions. The interaction of HeLa cellular proteins with the stem-loop structure formed by the RNA of the transactivating region (+ 1 to +62) of the human immunodeficiency virus has been studied with uranyl acetate using gel retardation as an additional criterion of specific binding (61). Rather than exhibiting a pattern of protection characteristic of protein-DNA interactions, enhanced cleavages or "imprints" diagnostic of binding are observed. OP-Cu yields parallel results with different preferred scission sitf:s. Although the specific binding of protein can be inferred using these experiments, the nucleotides bound by the protein cannot be deduced until the chemistry of the cleavage by these reagents is better understood. The application of ferrous-EDTA and MPE as footprinting reagents for RNA has not been reported.

222

SIGMAN & CHEN


SITE-SPECIFIC NUCLEASES The development of site-specific nucleolytic agents has been an active area of research in the past several years . There are two features in their design. The first is the ligand that will target the cleavage reaction. The second is the chemical method used for the scission of the phosphodiester backbone of the nucleic acid. Site-specific nuclease activities have been prepared using DNA binding drugs (2, 62; J. F. Constant et aI, in preparation), oligonucleotides (63-67) , proteins (68), and peptides (69) to target the cutting. The two most widely used nucleolytic activities have been 1, l O-phenanthroline-copper and ferrous-EDTA. Recently, a second copper-dependent oxidative system has been explored in which the metal is linked to the carrier via the tripeptide Gly-Gly-His (70). This tripeptide is responsible for the high-affinity binding of cupric ion to bovine serum albumin (71, 72). Initial attempts at cleaving DNA by metal ion-catalyzed hydrolysis have been made, but the efficiency of scission is orders of magnitude less than that obtained by oxidative chemistry (73 ). The goals of this research are several . The first obvious outcome would be a family of synthetic restriction enzymes with tailored specificity . These molecules might prove useful in the analysis of complex genomes. The second would be to enhance the ability of an antisense oligonucleotide to block gene expression by the attachment of a nucleolytic activity. These chemically reactive oligonucleotides might prove useful for research in cell biol ogy and possibly as pharmacological agents . Finally, nucleolytic activi ties attached to carrier ligands provide a unique method for exploring the binding specificity of the carriers themselves. DNA-Binding Drugs as Targeting Reagents The first site-specific nuclease activity was reported by Dervan and col leagues . In this initial set of experiments, they linked ferrous-EDTA to distamycin, an A-T-specific ligand that binds in the minor groove (62) (Fig ure 9) . Scission was observed in A-T-rich regions of a restriction frag ment. Longer analogs have been synthesized that yield enhanced specifi city. Hoechst dye 33258, a fluorescent ligand that binds in the minor groove at A-T-rich regions (74), has been alkylated with 5 -iodoacetyl-l, 1O phenanthroline. The resulting product is an efficient scission reagent with different scission specificity than any 5-substituted phenanthroline (J. F . Constant e t a I , in preparation) (Figure 9) . A s anticipated from the binding specificity of Hoechst 33258, the primary sites of scission are adjacent to A-T-rich regions.

CHEMICAL NUCLEASES


a

Figure9

223

b

Structures of synthetic scission reagents derived from (a) distamycin and (b) Hoechst

33258.

Polynucleotides as Targeting Ligands Oligonucleotides have been the most widely used targeting ligands with both ferrous-EDTA and 1 , 1 0phenanthroline-copper serving as the associated nucleolytic agent. Two dif ferent strategies were initially used for the synthesis of ferrous-EDTA-linked oligonucleotides. In the first (Figure 10), EDTA was linked to the 5' phosphorylated end of the carrier deoxyoligonucleotide by coupling an ethylenediamine linker arm to the 5' terminal phosphate using a water-soluble carbodiimide and then acylating it with the anhydride of EDTA (65) . In the initial experiments, a 1 6mer corresponding to sequence positions 7 to 22 of a target sequence was annealed and then Fe 2+ and dithiothreitol were added. A strong set of cutting sites were observed from positions 24-28 with a max imum intensity at position 26. There were a second set of cutting sites at positions 1 6- 18 (Figure 1 0) . Extensive nonspecific degradation was observed unless poly A was added as radical trap for oxidative species generated in solution . Flexibility of the linker arm as well as the diffusibility of the reactive intermediatl� could account for the 13-base span of cutting sites. A synthetic approach was used to incorporate a T modified with EDTA (T*) in the center of a 1 9-nucleotide sequence 5' -TAACGCAGT*CA GGCACCGT-3 ' (66) (Figure 1 1). Sixteen scission sites in the target DNA were observed about the modified T-residue when the reaction was carried out with 0. 5 J.1M 19mer and 1 nM target fragment. Carrier DNA was not WATSON-CRICK HYBRIDIZATION

224

SIGMAN & CHEN

�

- IMIDAlOLE.CDI� OLIGONUCLEOTID E OLIGONUCLEOTIDE - 5'-0 -0 0_

ETHYLENEDIAMINE..

o " 0L IGONUCLEOTIOE - 5' - 0- � - NH-CH2-CH2 - NH2

-

�O

' 5 -0- -

0_

EDTA ANHYDRIDE �


0_

30 I

37 I

3

I -

20 I

10 I

"�!!ll,, ,'l,

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5' EDTA - CACAA TTCCACACAAC- 3'

5'

-

I to

Figure 10 terminus

Oligonucleotide-directed scission

of

I 16

ferrous-EDTA using derivatization at the

5'

(65). (Top) coupling chemistry; (haltom) scission pattern.

reported to have been added. The extent of scission for the two examples of ferrous-EDTA targeting using Watson-Crick hybridization did not exceed 30%. The first reported example of the targeting of OP-Cu scission used a 21 mer corresponding to + 1 to +2 1 of the template strand of lac operon (63) as a carrier. 5-Glycylamido-l, IO-phenanthroline was linked to the 5' -phosphory lated terminus of a synthetic oligonucleotide using the water-soluble carbo diimidc method (e . g. Figure 10) (63). After annealing 2 /-tM of the OP oligonculeotide with 45 nM of the target strand, scission was observed from sequence positions +20 to +25. It was not necessary to add carrier DNA to block nonspecific scission. The OP-Cu scission reaction exhibits a tighter envelope of cleavage sites than ferrous-EDTA with less background cutting. If the same OP-Cu-linked oligonucleotide is compared as a scission reagent for DNA and RNA of identical sequence, parallel digestion patterns are obtained (64). In early studies with the nucleolytic activity of I, lO-phenanthroline-


CHEMICAL NUCLEASES

3

,

ttd ! �lttd ! jlh

- TA TTCGHATTACGCCATCAAATAG TG TCAATTT AACGA TTGC GTeAGTCeG TGGC AeATAC TTl AGAT TGT T At * , , 5 -TAACGCAGTCAGGCACCGT- 3

Figure II

225

- 5'

Oligonucleotide-directed scission of ferrous EDT A using internal derivation (66).

(Top) T* EDTA derivative of thymidine; (bottom) scission pattern.

copper, the 2: 1 1 , 1O-phenanthroline-copper complex was demonstrated to be the reactive species in the scission reaction (18). The 1 : 1 complex was ineffective as a scission reagent even if the coreactant hydrogen peroxide was added. The results with the deoxyoligonucleotide targeting of OP-Cu cleav age suggest that the second phenanthroline ligand is not required for the formation of the oxidative species resulting from the oxidation of the cuprous complex by hydrogen peroxide. Possibly, the second phenanthroline is es sential for Ithe stability of the noncovalent complex formed between the parent reagent and DNA. a-DEOXYOLlGONUCLEOTlDES P-linked deoxyoligonucleotides prepared from a nucleotides provide an approach for determination of the hybridization pattern of these abiological molecules. Since these deoxyoligonucleotides are resistant to hydrolysis by nucleases, they are potentially useful as antisense reagents. Using the 27mer 5' -TGAGTGAGTAAAAAAAATGAGTGCCAA3' as the target, the reactivities of OP-linked a-T8 and {3-T8 deoxyoligonucle otides were compared (67). The scission pattern indicates that the a-deoxyoli gonucleotide forms a duplex with parallel strands (Figure

12).

Triple-helix formation provides an attractive alternative to Watson-Crick double-strand hybridization for achieving sequence-specific scission of double-stranded DNA using oligonucleotides as carriers (75, 76). Triple helices form spontaneously and do not require the denaturation of the parent duplex. Triplex structures can form with poly U TRIPLE-HELIX FORMATION

226

SIGMAN & CHEN


[PJ

(51) (31) (OP)-TTTTTTTT

[P] [a]

(OP) -TTTTTTTT (31) (51)

Figure 12 a-deoxyoligonucleotides as targeting reagent for OP-Cu (67). Comparison of scis sion patterns produced by a- and {3-deoxyoligonucleotides in a complementary sequence.

and poly A or poly T and poly dA with a stoichiometry of 2-homopyrimidine and I -homopurine strands (77-79) (Figure 13). In the proposed structures , a homopurine and homopyrimidine form an A-helix with Watson-Crick base pairing. The second polypyrimidine binds via Hoogsteen-base pair formation to the purine strand with the same polarity as this strand. Comparable structures have been suggested for G and C; protonation of the C strand is required for stable Hoogsteen base-pairing in the major groove. The basic features of the postulated triple helix structure, proposed from fiber diffrac tion data, have been confirmed by high-resolution NMR studies using d(G A)4 and d(T-C)4 (78, 79). An intriguing feature of this work has been the demonstration of a dangling 3' C residue suggesting that the A-T Hoogsteen base pair is significantly more stable than the G-C Hoogsteen base pair in triple helices (78). Derivation of homopyrimidine strands with either ferrous-EDTA or 1,10phenanthroline-copper has permitted a chemical verification of the proposed structures and has suggested a potentially powerful new approach in the design of site-specific nucleases (75). The initial report made use of the synthetic target duplex presented in Figure 14 that was hybridized to (dT)15 derivatives that bore the modified thymidine derivative (T*) (Figure 11) either at positions 1 , 5, or 10. With the I T* derivative, the observed 5' staggered scission sites are diagnostic of major groove attack of the ferrous-EDTA as well as the parallel orientation of the Hoogsteen base-paired second pyrimi-

CHEMICAL NUCLEASES

227


c*

Figure 13

Patterns of hydrogen bonding proposed for triple helices (78). C* refers to protonated

cytosine. which binds to the G-C base pair.

dine strand relative to the purine strand. If the

5- or lO-T* derivative is used,

scission is observed exclusively on the pyrimidine strand.

(40 volume per 100 fLM of calf thymus DNA, 0.67 fLM DNA-EDTA probe, 25 fLM Fe2+, and 1 mM DTT. Cationic spennine or hexamine cobalt is essential to Reaction mixtures contain 1 mM spermine, ethylene glycol

cent),

minimize electrostatic repulsion between the three strands. Scission is not observed in the absence of spermine even in the presence of

8 mM magnesium

ion. The presence of organic solvents (e.g. ethylene glycol, methanol, etha nol, dioxane, or DMF) contribute significantly to the reaction, possibly by enhancing the stability of the A-helix. Sequence-specific scission mediated through triple helix formation has also

been observed using 1, lO-phenanthroline-copper as the nucleolytic agent (Figure 15)

(93). Although these results also demonstrate that the pyrimidine

strand has the same polarity as the purine strand, the scission sites are staggered in the

3' direction as would be expected from oxidative attack

originating from the minor groove. Since a prerequisite for cleavage is a chain length of the linker arm of

6, an intriguing explanation for the minor groove

attack is that the 1, lO-phenanthroline must traverse the stacked bases to approach the C-I hydrogen of the deoxyribose for productive oxidative attack. Initial attempts to develop triple helix formation as a strategy for chromo-

1 5 - CCCCCCCCCCAAAAAAAAAAAAAAAGGGGGGGGGG - 31 1 31- GGGGGGGGGGTTTTTTTTTTTTTTTCCCCCCCCCC- 5 Figure 14 sites.

Duplex DNA used to form triple helix with (dT),s (75). Bold face indicates scission


228

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I

SIGMAN

&

CHEN

..

1

.

TeeT GATIA A A G GAGGAG ilT GAAGAsT GA 3 IAG GACTA ! ! � � ! � � ! � ACTT CTsAC T

�

III··

�

31 5

I

,.,TTTeeTeeT CT

OP51

31

Figure 15 Duplex DNA and deoxyoligonucleotide used for targeted scission by OP-Cu via triple helix formation (J. C. Francois et aI, personal communication).

somal mapping has provided preliminary information on this form of hybridization (76). Studies of the stringency of the hybridization have shown that a one-base mismatch in a 15mer is sufficient to destabilize binding at 25°C. Using ferrous-EDTA-linked reagents, double-stranded breaks in DNA have been demonstrated-a requirement in the analysis of high-molecular weight DNA. If a reagent is to be used for chromosomal mapping, scission also must proceed within the agarose gel matrix in order to utilize pulsed-field gel electrophoresis to separate large DNA fragments. By synthesizing an EDTA-Fe-oligonucleotide capable of forming a triple helix with 5' A4GA6A4GA-3', a unique sequence occurring within the bacteriophage A genome, the double-strand scission of lambda with 25% efficiency has been achieved within 1% low melting agarose. The failure of this reaction and other site-specific scission reactions to go to

completion may be due to self-destruction of the reagent by the oxidative species generated. Incomplete cutting will result if the inactivated reagent binds tightly and cannot be displaced by competent reagent. Extensive use of triple helices will be dependent on the stringency of the hybridization reaction and the availability of sequences that are potential targets.


CHEMICAL NUCLEASES

229

R-LOOPS A new method for the sequence-specific scission of chromosomal DNA uses H.-loop formation for targeting and 1 , l O-phenanthroline-copper as the nucleolytic agent (94). R-Ioops form when RNA is hybridized to double stranded DNA in 70% formamide displacing the DNA strand of identical sequence (80). Recognition involves Watson-Crick base pairing and is not affected by cytosine methylation. Once formed , R-Ioops are stable in the absence of formamide , and can be visualized in electron microscopy, purified by gel filtration, and digested by restriction enzymes (8 1, 82). The advantage of this approach is the potential of cleaving at any desired sequence. Homo purine/homopyrimidine stretches are not required. The scission reagent is prepared by cloning the selected sequence into an RNA expression vector utilizing T7 RNA polymerase. In vitro transcription is carricd out in the usual way, except that 5-allylamine UTP is used in place of UTP. The n�suIting transcript is then modified at the 5-allylamine group and used to fonn an R-Ioop with the target sequence (Figure 1 6) . Following activation with cupric ion and 3-mercaptopropionic acid , single-stranded nicks within the region of hybridization are observed on the complementary strand as well as the displaced strand when denaturing gels are used to assay the products of the reaction. When nondenaturing gels are used to analyze the scission products, double-stranded nicks are evident . R-Ioop-directed scission is currently being evaluated with larger DNAs in order to determine if it provides a competitive alternative for the analysis of complex genomes. Although agaroses are available that are resistant to high concentrations of formamide, the efficiency of R-Ioop formation within a gel matrix is un known .

DNA-Binding Proteins as Affinity Ligands DNA-binding proteins such as repressors and transcription factors recognize sequences that are at least eight base pairs long and therefore are potentially powerful vehicles for targeting nucleolytic activities. Since they bind to double-stranded DNA, they share the advantage of triple helices in not requiring denaturation of the target duplex. The first DNA-binding protein that was chemically converted to a site-specific nuclease was the E. coli trp repressor (68) which, with L-tryptophan as a corepressor, binds tightly to three E. coli operators-aro H, Trp EDCBA, and Trp R (83-86). The method of derivatization of the protein is straightforward and involves covalently attaching the 1 , 1 O-phenanthroline to Iysines via an iminothiolane linker. For E. coli trp repressor, which contains four Iysyl residues, four 1 , 1 0-phenanthroline derivatives were incorporated. However, since reaction at the amino-terminus has not been excluded, mUltiple modified forms of the repressor may be present . The positions of the Iysyl residues in the trp

230

SIGMAN & CHEN

1' 0 cloned gene

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b. Spin Column; RNose; phenol extract

• A

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3' 5' 3'

Figure 16

8

+ 6

3'

5' 3' 5'

Strategy for sequence-specific scission using R-loops (94).

repressor relative to DNA based on the three-dimensional model of the protein provided by Sigler and colleagues are presented in Figure 17 (87, 88). DNase I footprinting has demonstrated that the trp repressor exhaustively modified with 1 , l O-phenanthroline binds to the aro H operator with compar able affinity as the unmodified repressor. Upon the addition of cupric ion and thiol, scission is observed in the sequence positions that comprise the pro tected domain of the fingerprint. This cutting is dependent on the corepressor

CHEMICAL NUCLEASES

23 1


39

29

Figure 1 7 (87)] .

Positions of lysyl residues in 3-D structure of E. coli Trp repressor [adapted from

tryptophan. Assay of scission on both strands reveals a 3 ' stagger characteris tic of minor groove scission. Since the protein binds to the major groove and would presumably block the approach of any oxidative species, the scission could only be observed if the oxidation proceeds in the minor groove. Extensive clltting is observed upon prolonged incubation of the trp EDCBA operator (Figure 18) . After 20 hours, single-strand nicks are observed in 50% of the labeled strands . S ince double-strand scission is also observed (although at a reduced yield), the modified repressor can cycle while bound to the DNA and therefore acts catalytically. The flexibility and length of the lysyl residue with the iminothiolane linker may allow 1, lO-phenanthroline to both approach the minor groove and extend in solution for reduction by the thio!. As with the other reagents, the efficiency of the reaction must be improved and sources of background cutting, such as low-affinity nonspecific binding of the protein, suppressed. The 14-bp recognition sequence of the repressor presents an additional problem of this approach. Although the repressor binds three homologous but nonidentical operators-Trp R, Trp EDCBA, and aro H-the binding motif may be too rare to be useful. However, the E. coli trp repressor with new specificities has been isolated using the "phage challenge method" pioneered by Youderian and colleagues (89, 90). Proteins of diverse specificity may be isolatable by this intriguing procedure. The chemical conversion of the trp repressor is of interest in a different

232

SIGMAN

&

- trp

CHEN 0.5

4

20


2

Hours

to

e ®

a

b

c

d

e

f

9

Figure 18 Scission of TrpEDCBA operator with E. coli trp repressor derivatized with 1 , 10phenanthroline (68). Lane a: OP-Trp repressor with Cu2+ and 3-mercaptopropionic acid but no L-tryptophan, incubation time 4 hours; lane b: DNase I footprint of OP-trp repressor with L-tryptophan; lanes c to g; OP-trp repressor with L-tryptophan, Cu2+ , and 3-mercaptopropionic acid incubated for 0.5, I , 2, 4, and 20 hours , respectively at 37° C.


CHEMICAL NUCLEASES

233

context. Attachment of nucleolytic agents to carrier ligands provides a chemi cal method to probe the stereochemistry of the binding of a ligand. For example, the scission pattern observed upon the hybridization of OP-Cu linked a deoxyoligonucleotides to a target sequence revealed the duplex was composed of parallel strands. Similarly, the orientation of the second pyrimi dine strand participating in Hoogsteen base pair formation in the major groove was shown to adopt a parallel orientation relative to the purine strand using deoxyoligonucleotides coupled to either Fe-EDTA or OP-Cu. The structure of the trp-repressor-operator complex has now been solved and has predicted nucleic acid-protein interactions that have not been anticipated from previous studies (88) . This binding model can be tested with the chemically modified trp represso:r once the observed scission sites can be associated with specific Iysyl residues.

Chemically Synthesized Peptides for Directing Scission

Peptides can also serve as carriers of a DNA scission activity. The first reported example was the 52-amino-acid peptide derived from the C terminus of Hin recombinase, an enzyme that alters the expression of flagellin genes of Salmonella typhimurium by inverting a DNA segment (9 1 ) . The 52-unit peptide includes a sequence of 2 1 amino acids with homology to the helix tum-helix format of known repressors. By means of a DNase I footprinting assay, it was possible to demonstrate that the 52-unit peptide containing this helix-turn-helix format bound to the same hix binding site as the parent recombinase . However, a 3 1 -unit peptide containing the helix-turn-helix format did 1I10t protect against DNase I digestion in a footprinting· assay. Attachment of ferrous-EDTA to the amino-terminus of this 52-unit peptide generated site-specific scission from the minor groove (69) . Double-strand scission was also observed with the plasmid containing the Hix L site. However, alternate scission sites are observed even when nonradioactive random sequence DNA is added to the reaction mixture to absorb reactive intermediates generated in solution. The tripeptide Gly-Gly-His has also been added to the amino-terminus of the peptide derived from hin recombinase (70). This tripeptide has high affinity for cupric ion. Since the free complex can accomplish oxidative DNA scission in solution (92), the tripeptide-cupric complex can also serve as the nucleolytic agent when attached to the amino-terminus of the 52-unit peptide. The scission pattern obtained with this peptide has a narrower envelope of bands compared to the same peptide with ferrous-EDTA on the amino terminus. OP-Cu also shows a more restricted pattern of cutting that suggests that the two copper-based systems may be generating the same oxidative species. The: tripeptide has intriguing potential because it may allow the


234

SIGMAN & CHEN

incorporation of a nucleolytic agent by protein or peptide synthesis without resort to chemical modification. In general , an important constraint in using peptides for targeting is their intrinsic confonnational stability . Preliminary results with an OP-Cu 2 1 amino-acid peptide derived from the helix-turn-helix motif o f the trp repressor indicates that preferential cleavage can be observed at the same sequence positions as the intact protein (C . -h. B . Chen, D. S . Sigman, in preparation) . However, extensive background cleavage is observed because of the large excess of random coil peptide derivatized with OP-Cu in solution. Unlike the 52-unit peptide derived from the hin recombinase, the binding of this peptide cannot be detected using DNase I footprinting. CONCLUSIONS/FUTURE DIRECTIONS Chemical nucleases currently provide a viable alternative to more widely used footprinting techniques using DNase I and dimethyl sulfate. Combination of footprinting with gel retardation provides a new methodology that enhances the power of each rocedure. The other aspects of chemical nucleases re viewed have promise but require further development. None of the site specific nucleases are yet sufficiently efficient or specific to challenge seriously restriction enzymes as reagents to manipulate and analyze DNA. More extensive analysis of the underlying chemistry of scission by the various reagents will be required before they become a definitive method for the analysis of structural variability.

p

ACKNOWLEDGMENTS

We acknowledge useful conversations with members of our research group as well as Henri Buc and Annick Spassky of the Pasteur Institute , and Richard Gaynor and Randolph Wall of UCLA. Research in our laboratory has been supported by the National Institutes of Health and the Office of Naval Research. Literature Cited I . Sigman, D. S . , Graham, D. R . , D 'Aurora, V . , Stem, A . M . 1 979 . .r. Bioi. Chem. 254 : 1 2269-72 2. Hertzberg , R. P. , Dervan, P. B. 1982. J. Am. Chem. Soc. 1 04: 3 1 3- 1 5 3 . Tullius, T . D . , Dombroski, B. A . 1986. Proc. Natl. Acad. Sci. USA 83:5469-73 4 . W ard , B . , Skorobogaty, A . , Dabro wiak, J. C. 1 986 . Biochemistry 25: 6875-83 5 . Le Doan, T . , Perrouault, L. , Helene, c . , Chassignol , M . , Thuong, N. T. 1 986 . Biochemistry 25;6736--39

6. Nielsen, P. E . , Jeppesen, c. , Buchardt, O. 1 988. FEBS Leu. 235 : 1 22-24 7. Barton , J. K. 1 986. Science 233:727-34 8 . Stubbe, J . , Kozarich, J. W. 1 987. Chem. Rev. 87: 1 107-36 9. Goldberg, I. H . 1 987. Free Radic. Bi oi. Med. 3:41-54 1 0 . Zein, N . , Sinha, A. M . , McGahren, W . J . , Ellestad, G . A. 1 988. Science 240: 1 1 98-201 1 1 . Chowdhry, V. , Westheimer, F. H . 1 979. Annu. Rev. Biochem. 48:293-325 1 2 . Tullius, T. D. 1 988. Nature 332:663-64


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51

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