157

Biochimica et Biophysica Acta, 4 4 2 ( 1 9 7 6 ) 197--207

© ElsevierScientific PublishingCompany, Amsterdam.-Printed in Th~ Net~'~ands

BBA 98665

FURTHER PURIFICATION AND CHARACTERIZATION OF I"4 ENDONUCLEASE V

SEIICHI YASUDA* and MUTSUOSEKIGUCH! Department of Biology, Faculty of Science, Kyushu Univer~yo F u k ~ k a 8 ~ ( J ~

(Received December23rd, 1975) (Revised April 21st, 1 9 7 6 ) Summary

T4 endonuclease V, which is involved in repairof ultmviolctrocedure 2. This assay measures the liberationof phosphomonoe~ter ~roups produced by incision of D N A . The ~eaction mixture {0.25 ml} cont~med 16 nmol ~:P-labeled, dephosphorylated and ultrav~olet-hvad~a~ed {840 J~m:} T 4 D N A , 10 #mol Tris. HCI (pH 7.5), 2.5 ~mol MgCl: and enzyme. [ncut~t~on was at 37°C for 10 rain after which 0.05 ml calf thymus D N A {2 m g h r ~ ~ad 0.03 ml 50% trichloroaceticacid were added and the mixture was ce~ttr~f~tged at 1000 X g for 10 rain in the cold. The precipitatewas d~ssch~d in 0.1 ml 0.1 N N a O H and neutralized with 0.01 ml 2 M Trls- HCI (pH 5.7}.E. cot~ ~kal~ae phosphatase and E D T A were added tv finalconcentrations of 6 uni~s~ml and 5 m M , respectively.This mixture (0.25 ml) was incubated at 45°C for 30 m~n. At the end of incubation 0.1 ml calf thymus D N A , 0.05 ml I ~! MgCl~ ar~ 0.04 ml 50% trichloroaceticacid were added in succession. After c e n ~ r i I ~ ~t 1000 × g for 10 rain in the cold, 0.1 mi of 5 % charcoal suspension w~s added to ~the acid-soluble fraction and stood at room temperature for 5 rain with occa~onal shaking. The mixture was centrifuged, and the super~atant fluk~ ~ s L~eated with charcoal once more. The radioactivity in the final supernatant solution was determined. The activity is proportional to enzyme concentration until about 40 pmol phosphate is converted to the phosphatase~sens~ti~ ~orm. Background level varied from 3 to 10 pmol, depending on the prep~rat~on of dephosphorylated 3~P-labeled D N A . One unit of the enzyme is defined as the amount catalyzing the conversion of 1 nmol **~Pfrom ~rrad~ated D N A into phosphomonoesterase-se~itive forms in 60 rain under the standard co~lit~ons, and this almost corresponds to one unit of the activitydefined in P r o ~ u z e 1.

200

Other methods Preparation of DNA.cellulose. DNA-cifllulose was prepared by the procedure of Alberts et al. [18]. 15 ml ofE. coli DNA (1.5 mg/ml), either irradiated with a 15 W germicidal lamp (10 rain at a distance of 10 em from the lamp) or not, was mixed with 5 g of cellulose powder (Munkte1410). The mixture was dried at 42°C overnight, washed with ethanol and 1 mM NaC1 and dried in vacuo. Determination of protein. Protein was determined by the method of Lowry et al. [19]. Results

Appearance of endonuclease V after T4 infection Fig. 1 shows the time-course of appearance of T4 endonuclease V in phageinfected cells. The enzyme activity increases rapidly after infection with T4D and reaches its maximum level within 10 min after infection. Thus, endonucleas• V seems to be one of the early enzymes induced b y T4. No increase in the enzyme activity was induced by infection with T4v,. It has been sho~m that early enzymes of T4 are overproduced when phage DNA synthesis is inhibited by amber mutation or ultraviolet irradiation [20,21]. In an attempt to obtain extracts with higher specific activity, cells were infected with T4 amN82 (gene 44) or ultraviolet-irradiated T4 wild type. In neither case was the level of endonuclease V greater than in normal infection (Fig. 1). It seems that control of the formation of endonuclease V differs from that of other early enzymes.

Purification of endonuclease V In the purification procedure all operations were carried out at 0--4°C and endonuclease V was amayed with Procedure 1. A typical purification is summarized in Table I. Preparation of extract. E. coli 1100 cells were grown in broth at 37°C with

of 0

?

4_

~

10 20 30 Time after infection (rnin)

Fig, I . Appearance of endonuelease V after phage infection. Log-Phase culture of E, coli 1100-14 (End I ' , s u ' ) was infected w i t h T 4 D , T 4 a m N 8 2 , ult~avlolet-hTadlated ( 5 4 J~m ~) T 4 D or T 4 e z at a raultiplicity of 5. A t the times ahown Portions were w ith d raw n a nd chilled quickly w i t h crushed ice. ExtnLcts were prepared as de$cril:ed in the section on purification. Enzyme activ/ty was determined b y Procedure I with 1 0 0 ~g protein/rnl of extracts. • • , infection, w i t h T4D~ c o, infe c tion with T 4 arnN82; X X, infection with ultraviole t-in-adiated T 4 D ; ~ ~. infection w i t h T 4 v I.

TABLE I P U R I F I C A T I O N O F E N D O N U C L E A S E V F R O M T 4 D . I N F E C T ~ D E. COL1 1 ~ 0 0 Fraction

To~al activity (u~ts)

Yield

3500 7200 * 930 1040 830 580

100 207 27 30 24 17

(%)

S~.~© ac~vit~

(t~tts~mg p~ot~n)

I n IU IV V VI

Extract Phase p a r titio n eM-Sephadex 1st Hyd~rcxyapatite 2nd Hy ~ o x y a ~ t i t e I r r a d t a t ~ i DNA-eellulose

0.5~ ~.11 244 579 867 2120

1 411 97~ 1~61 3573

* T o t a l activity in Fraction II always exceeds t h a t o f extract. TIKs is n o t due to e ~ m ~ a ~ o f iml~bitors p ~ s e n t in extract, since excess of a n extl~ct o f T 4 r l d n f e c t e d ceL~s is ~ c l ~ in ~ r e ~ ~tion mixture. It may be due to aeti~ration b y p o l y e t h y ~ n e glycol or de~ttt~u,

aeration to a concentration of 5 • 10 s cells/ral and infected with T4 at a ~nultiplicity of 5. At 15 min after infection the culture was chilled rapidly by ~our. ing onto crushed ice. Cells were collected in a refrige~ated cen~ifuge ar~l cell paste was stored at --20°C. 20 g T4-infected cells were suspended in 80 ral 0.01 M Tris. HCI (pH 7.5) containing 1 raM 2-raereaptoethanol and 10% eth~ lene glycol and disrupted with an ultrasonic disintegrator (K~djo Electric Co.). The lysate was centrifuged at 10 000 X g for 20 rain and the supernatant fluki (9~) ral) was taken as an extract. Phase partition. To 90 ml extract were added 10.3 ml 20% (w/w) dex ~ n 500, 28.9 ral 30% (w/w) polyethylene glycol 6000 and 15.1 g NaCI with ~ontinuous stirring. After additional stirring for 60 rain, the mixture was eel trifuged at 1000 × g for 10 min and the clear upper layer (110 ral) was ~ e n (Fraction II).

CM-Sephadex chromatography. Fraction II was dialyzed against two chm~:~ of 3 l 0.01 M potassium phosphate (pH 6.5) containing 0.01 M 2-raercap oethanol, 10% ethylene glycol and 2 m M E D T A and applied to a column of Ct~Sephadex C25 (1 cra X 15 cra) equilibrated with the same buffer. After wm~ing the column with 20 ral of the buffer, 200 ral of the buffer containing a linear gradient (0--0.5 M) of KCI was applied. The flow rate was 6 ralfn and ,~ral fractions were collected. The enzyme activity appeared after passage of 10 .~ ral of the gradient solution. The active fractions (50 ral) were pooled (Fractiol ]I]). Hydroxyapatite chromatography. Fraction HI was dialyzed against 2 l 0.0~ M potassium phosphate (pH 6.8) containing 0.01 M 2-raercaptoethanol, I0~ ethylene glycol and 2 m M E D T A and applied to a column of hyclroxyapatR~ (Hypatite C, 1 cm X 15 cm) that had been equilibrated with 0.01 M potassium phosphate (pH 6.8) containing 0.01 M 2-mercaptoethanol and 10% ethylene glycol. The column was washed with 20 ml of the equilibrating buffer, followed by a linear gradient of 200 ral of potassium phosphate (pH 6.8) with limits of 0.01 M and 0.4 M, containing 0.01 M 2-mereaptoethanol and 1 ~ ethylene glycol. The flow rate was 6 ml/h and 5-ml fractions were collected, Enzyme activity appeared after pamage of 100 ral of the gradient. Active fractions were

202

pooled (50 ml, Fraction IV) and dialyzed againsttwo changes of 210.01 M po. tassium phosphate (pH 6.8) containing 0.01 M 2.mercaptoethanol, 1 0 % ethylene glycol and 2 m M E D T A . Dialyzed Fraction IV was then applied to a small hydroxyapatite column (0.7 crn × 6.5 cm) which had been washed with 0.01 M potassium phosphate buffer (pH 6.8) containing 0.01 M 2.mercaptoethanol and 10% ethylene glycol. Then a linear gradient of 80 ml potassium phosphate (pH 6.8) with limits of 0.01 M and 0.4 M, containing 0.01 M 2-mercaptoethanol and 10% ethylene glycol was applied. The flow rate was 2.5 ml/h. Fractions with specificactivity greater than 100 units/mg protein were pooled and dialyzed against 0.04 M Tris • HCI (pH 7.5) containing 0.01 M 2-mercaptoethanol, 10% ethylene glycol and 2 m M EDTA. This fraction (Fraction V, 22 ml) was stored at 0°C without loss of activity for more than 4 months and used in most of experiments described here. Irradiated DNA.c~llulose chromatography. 8 ml Fraction V was applied to a column (0.7 cm X 5.7 cm) of ultraviolet-irradiatedDNA-cellulose, pre.equilibrated with 0.04 M Tris • HCI (pH 7.5) containing 0.01 M 2-mercaptoethanol, 10% ethylene glycol and 2 m M E D T A . After washing the column with 4 ml of the equilibrating buffer containing 0.15 M NaCI, 20 ml of the same buffer containing a linear gradient of 0.15 M to 1.0 M NaCI was applied. Enzyme activity appeared after passage of 10 ml of the gradient. Active fractions (Fraction VI, 4 ml) were pooled and dialyzed against 1 ] 0.04 M Tris • HCI (pH 7.5) containing 0.01 M 2-mercaptoethanol, 10% ethylene glycol and 2 m M E D T A . The enzyme was purified 3600 fold over the crude extract. Fraction VI contained a trace of nuclease activity which degrades D N A alkylated by methyl methanesulfonate. This contaminating activity can be removed by chromatography on a non-irradiated DNA-cellulose column, details of which are described in the accompanying paper [22]. Conditions for the reaction The activity of endonuclease V in the purified fraction was assayed under various conditions. Procedure 2, which measures formation of alkaline phosphatase.sensitivephosphates, was used in these experiment~. First,reaction was performed in Tris • HCI or Tris. maleate buffer at various p H values. It was found that endonuclease V is most active between p H 7.(}and 7.5. About 50% of the m a x i m u m activity(pH 7.2) was obtained at either p H 6.4 or 8.1. Endonuclease V is active in the absence of divalent cations and activated 1.5to 2-fold by any one of CaCl2, MgCl2 and MnCl2 at concentrations of 2--5 raM. In addition, it was found that the enzyme is activated by 50 m M NaCI or KCI. These results suggested that endonuclease V is activated by high ionic strength rather than by specific ions. This was confirmed by the finding that the rate of reaction in the presence of 5 m M E D T A is 1.7 fold of that in the absence of EDTA. Kinetics of the reaction Fig. 2 shows the time course of the reaction under the standard assay conditions (Procedure 2). The reaction proceeds linearly for about 15 min and then levels off. Addition of the enzyme at 30 rain has no effect,indicatingthat the

cessation of the reaction is not due to inactivation of endonuel~se V. The f m ~ extent of the a~p release is about 55 pmol. A similar result was obtained when in'adiated D N A was incubated with ~fious m o u n t s of endonuclease V. Within certain enzyme concentrations, the amount of 3~p released was proportional to the amount of enzyme used, but no more than 53 pmol 3~p was released even though a large amount of enzyme w ~ used. The D N A used in these experiments contained 72 pmol of p ~ n k ] i n e dimers and it seems that the reaction is limited by the number of dime's. Next we investigated the action of endonuclease V on n~ive and denatured D N A . W h e n T4 D N A was irradiated with varied doses of ul~aviol~t, he~t der~tured and treated with endonuclease V (Fraction VI), the number of phv~ph~tase-sensitive phosphates increased with increasing doses of ultraviolet, However, the rate and the extent of the reaction with denatured irradiated D N A w e ~ less than with non-heated D N A irradiated with the same dose of uRravio]et. Nonirradiated D N A , irrespective of native or denatured, does not serve as substr~t~ for the enzyme. These results are consistent with the previous finding, which were obtained with less purified enzyme preparations [7]. Fig. 3 shows substrate saturation curves of endonuclease V for native and denatured D N A . The Lineweaver-Burk plot of the curves yielded a K m value of 2.25 • 10 "s M for both native and denatured D N A (Fig. 3 inset).

Inactivation of endonuclease V Although endonuclease V is relatively stable when stored at 0°C it is extremely heat labile and loses 8 0 % of its activity within 8 rain of p~e~ncu~fion

6C

o~ --

•

,~

~

~,

~

.

~

T~me o~ i~cubaUon (m~n)

~o

-~

o~

~0

2O

30

43

50

Fig. 2. T i m e course of the endonuclease V reaction. Condition~ werv the same ~ 1 ~ c ~ i ~ - e 2 . 0 , ~ u ~ of endonuclease V (Fr~ctlon V) was included and the reactinn was t~,n~inated a t varlou~ t l m ~ as ~1~eated in tl~e figure, Additional enzyme (0.23 unit) was added a t S0 rain (arrow) axld incul~L~d f ~ ~ m ~ . Fig, 3. Substrate satttt'~,~ion of e n d o n u c l e a ~ acUvlty, a:P-labe|~] T 4 D N A was L r r ~ l i a ~ a t a ~ c e af 1 0 em from a n ultraviolet lamp for 2 rain an d divided into two poxtions. One pc~'tion o f D N A was d ~ o tuz ~ l by heating a t 1 0 0 ° C for 1 0 min in 0 . 1 5 M NaCl and chUKug in ice. U l t ~ t ~ e d ~Uv-~ denatured DNA a t eoneenttatlons of 6.4~ 8 . 9 6 , 12.8, 2 5 . 6 and 6 4 . 0 ~M were ~ n e ~ t e d f o r 1 9 m ~ a t 37°C w i t h 0 . 1 4 u n i t of endonuelease V (Ftact/orl V) in 4 0 raM T r ~ - HC! {pH 7 , 5 ) / 1 0 m M MICI:. T e ~ minal phosphates were determined ~ in P1"oeedu~ 2. f r e t : IAneweave~-Burk plo~ o~ th~ ~ -• "-, native DNA" o o. denatured DNA.

204

at 40°C (Fig. 4). The pattern of inactivation ~ almost the same whether the substrate is native or denatured DNA,

Inhibition by p.chloromercuribenzoate The enzyme is inhibited by p-chloromercuribenzoate. When endonuclease V was assayed in the presence of 4 • 10 "5 M and 4 • 10 "4 M p-chloromercuribenzoate, it exhibits 40--50% and 20--30% of normal activity, respectively (Table II). It is noted that the inhibition is similar for both types of DNA, These results favor the assumption that the activities toward native and denatured DNA are due to a single enzyme but not to two enzymes with similar specificity.

(a)

~o

>SQ

6--I (c)

N

(b)

2T;me of4 heot;ng ~0 (m;n~~

0

/

/

~

I

(d)

10Time of 20 0 10 incubation (re;n)

20

Fig. 4. Heat inactivation o~ endonuclease V. Endonuclease V (Fraction V) was heated at 40~C for various periods indicated, and assayed under the standard conditions (Procedure 2). lffadiated native and denatured DNA were used as substrates. Initial activity of endonuclease V was 0.29 uuit per assay, ¢ ~, native DNA; o o, denatured DNA. Fig. S. Spleen phosphodiesterase digestion of endonuclease V-treatod DNA. Conditions for phosphodiesterase digestion was as follows; Reaction mixture (0.25 ml) contained 0.64 #tool endonuclease Vtreated 14C.labeled E. coU DNA, 10 pmoITris • rnaleate (pH 6.5) and 0.02 u n i t (a and b) or 0.2 u n i t (¢) and (d) of spleen phosphodiesteras¢. After incubating at 37"C for 10 and 20 rain 0.05 m l bovine serum albumin (2 mg/ml) and 0.03 ml 50% ~iehloroacetic acid were adde d. Radioactivity in the acid.soluble fraction was determined. Substrates for c o n t ~ l experiments (a and b) were prepared as desexibed in Experlmental Procedures. Endonuelease V-treated DHA (c) was prepased as follows: 32 n m o l of t4C-labeled E. eoU r,NA was irradiated with ultraviolet (S40 J / m =) and incubated with excess (1.1 units) of endonuclease V (!~'aetlon VI) in the presence of 40 ram "Iris • HCI (pH 7.5) and 10 mM MgCI~ at 37°C for 40 rain. DNA ~as preeip|tated with triehloroacet/c acid and dissolved in 0.02 M Trls • HCl (pH 8.0). One portion was treated with alkaline phosphatase as described in Experimental Procedures. Subatrate f o l the last experiment (d) was prepared in a similar mannar in the absence of e n d o n u e l e ~ V. (a) S'*hydroxylo and 5'phosphoryl-terminated DNA. (b) 3'-phoapholyl- and 5'-hydroxyl-terminated DNA, (e) endonuelease V~reated ultraviolet-irradiated DNA. (d) ultraviolet-irradiated DNA without e n d o n u c l e u e V treatmarlt. ee+ substrate was pret~eated with phosphatase; o 0, subatzate without phosphatase pretreatment.

TABL~ II INHIBITION O F B N D O N U C L Z A S ~ : V BY p-CHLOROM~RCURm~NZOAT~ Endonucleu~ V (Fraction V) was dialyzed a ~

0.04 M T ~ • HC! ( ~

~ly¢ol a n d 2 m M F.DTA. Denatut'~d D N A w a s p ~ p ~ m ~ t ~a d @ ~ ~ ~. same as t h o ~ o f l ~ o c ~ l u ~ e 2 e x c e p t f o r a d d i t i o n o f ,~.e.J~l~om m ~ a a ~ m t e

[PCMB] (M X 10"s) 0 4 40

Relative actlvlty

7,5~ c o n ~ 3. O ~ ~

1~

~

~

~ ) .

(%)

NativeDNA

D~natt~lDNA

100

IOO

44

49

23

30

Analysis of 5°.terminiof endonuclea.se V-treatedD N A It is known that b o v i n e spleen phosphodiestera_~ degrades 5'-h)~roxyl-~minated polynucleotides exonucleolyticallyin the 5' -~ 3 °dh~ction and tl~ttits action is inhibited by the presence o; 5'.phosphate [23]. W h e n ultmviolat-irradiated~4C-labelad E. co~ D N A was t ~ t e d with m~ excess of endonuclaase V and then incubated with spleen phc~hod~tera~v with or without phosphatase pretreatment, endonuclaase V - ~ t e d D N A w~t~ d ~ graded rapidly only when it was pmtreated with alkalinep h o s p h ~ (F~. 5}. In this experiment ten times as m u c h phosphodiesteras~ as that us~d in the c~ntrol experiment with non-in'adiated D N A was added, s~nce the action of ph~sphodiesterase is partiallyprevented by pyzimidine dime~r [24]. This r~ult ~dicates that endonuclease V produces 5'-phospho~'l (and presumably 3'~hydroxyl) termini. Discussion T4 endonuclease V seems to be one of early enzymes inducvd by T4. The activityappears very rapidly in infected ce~.Isand reaches itsmaximtuu lavelwithin 10 rain after infection. However, unlike other emrly enzymes its synthes~ t~minates at the normal time even when infection is carfiod out with ult~olatirradiated phage or with mutants having a mutation in an essent~tlem~y function. It has been shown that the synthesis of early enz~ym~s continues beyond the normal time of cessation of enzyme formation whvn phage D N A s y n ~ is blocked by infection with such phages [20,21]. Thus, there is the L~sibility that formation of endonuclease V is regulated by a mechanism differentfrom that of other early enzymes. T 4 endonuclease V is unique in m a n y p r o ~ e s . It intz~du~s singla-strand breaks into ultraviolat-irradiatedD N A but h ~ no effect on native or denatured D N A . The enzyme reaction is a function of the ultravioletdose used for h1~idiation of the D N A , and the number of breaks formed almost c~rresponds to the number of dimers present in the D N A . The enzyme shows optimal activity ~t p H 7,2 and does not require added divalent ions. These properties are c o m m o n to those of irradiatedDNA-specific endonuclease isolated from M~crococcus lu-

206 teus and E. coli [10--13]. The molecular weight of T4 endonuclease V is approximately 18 000 and it is slightly larger than the enzymes of M. luteus (14 000--15 000) and o f E . coli (less than 14 000). It has been shown that T4 endonuclease V attacks both double- and singlestranded DNA provided that the DNA has been irradiated [7,9]. Data presented in this paper confirm this and show further that the activities on the two types of DNA are inactivated by heat or inhibited by p-chloromercuribenzoate in parallel fashion. It is likely that the activities are due to a single enzyme and n o t to two enzymes with similar specificity. This implies that endonuclease V recognizes the chemical structure of pyrirnidine dimer itself rather than the localized conformational distortion of the DNA molecule, caused by the formation of dimers. Substrate saturation experiments gave a Km value of 2.25 • 10 "s M for both native and denatured DNA, irradiated with ultraviolet. This value is comparable to that of the endonuclease of M. leuteus (1.25 • 10 -s M) [25]. The coincidence of gm values for both native and denatured DNA may be interpreted as follows: Endonuclease V binds to pyrimidine dimers in native and denatured DNA with an equal efficiency, but the succeeding step breaking phosphodiester bonds is faster in native DNA. We have shown previously that endonuclease V produces a break at a point to the 5'-side of a pyrimidine dimer [7]. It was proposed that a 5' -* 3' exonu. clease excises dimer-containing nucleotides from 5'-end to 3'- direction, followed by the gap.filling and joining reactions by DNA polymerase and ligase. In fact, an exonuclease activity was found in an extract of T4v~-infected cells which excises dimer-containing nucleotides from ultraviolet-irradiated, endonuclease V-treated DNA [27]. The activity was recently characterized as 5' -~ 3' exonuclease [28]. In the present study it was shown that the break produced by endonuclease V possesses a 3'-hydroxyl terminus. Evidence that the enzyme creates 3'-hydroxyl and 5'-phosphoryl termini was recently presented by Minton et ah [9]. The endonuclease of E. coli seems to produce the same termini since it is reported that the break is sealed by E. coil polynucleotide ligase which acts on breaks with 3'-hydroxyl and 5'-phosphoryl termini [13]. The 3'hydroxyl termini may be favorable for repair replication by DNA polymerase by serving directly as primers. On the other hand, it was demonstrated that the M. luteus enzyme produces 3'-phosphoryl and 5'-hydroxyl termini [23]. The biological importance of endonuclease V is evident. The activity is not found in an extract of cells infected with v mutant of T4 [4--9]. Recently, mutants with temperature-sensitive mutation in the v gene were isolated, which exhibit increased sensitivity to ultraviolet when plated at high temperature while exhibiting normal sensitivity at low temperature. Endonuclease V isolated from cells infected with such mutants is more thermolabile than is wild t y p e enzyme [29]. This indicates that the v÷ gene is indeed the structural gene for endonuclease V and that the enzyme is involved directly in repair of T4 DNA in vivo. Moreover, it was demonstrated that endonuclease V is capable of replacing the functions that are missing in uvr mutants of E. eoli or cells from patients with xeroderma pigraentosum, a human hereditary disease [30,31]. It seems that the first step of excision repair in many organisms is catalyzed b y endonuclease whose mode of action is similar to that of T4 endonuclease V.

~7 Acknowledgements W e thank Mr. K, Shimizu for stimulatingd~scu~on, T ~ study w ~ sug" pored by researchgrants from Ministry of Education, ScOnce ~nd ~ t u ~ of Japan and the Naito Research Grant for 1973. References 1 Straum, B., Searashi, T. and Robbins, M. (1965) ~ c . N a i l A ~ . SeL U,$~ ~ , g3Z--~3~ 2 G r o ~ a n , L., Kushner. S., Kap~n. J. ~n~l M~l~r, L (1969) C ~ t S ~ ~ S~p, ~ ~ 33, 229--234 3 Ta~agi, Y., Seklguchl, M., Okubo, S., Nakayama. H.. S ~ m ~ , ~., Y ~ , 8., N ~ @ I ~ , T~ Y o s h i h ~ , H. (1968) Cold Spring Harbor Syrup. Q u l n L Biol. 33, 219~227 4 Harm, W. (1963) Vlzo|oZy 19, 66--71 5 Katsukl, M. and Sek:Iguchl, M. (1975) Bioehlra. Biophys. A c ~ ~ 3 ~ I ~ 1 9 4 6 Yasuda, S. and Sekiguehi, M, (1970) J. MoL Biol. 47, 248~255 7 Yasuda, S. and 8eldgu©hl, M. (1970) Proe. Nal;L A ~ d . 8c|. U,~, 67,1839--184~ 8 Fricdberg~ E.C. and King, J.J. (1971) J. BacterioL 106, 5 0 ~ - ~ 7 9 Minton, K,, Durphy, M., Taylor, R. and Frt~Ibe~, E.C. (1~75) ,~. Biol, C 1 ~ . 2~), ~ 2 ~ - - ~ 10 Kaplan, J.C., Kushner, S.R, and Grossman, I,. (1969) P~oc, ~ U . A~.d. Sol. U,S, 63,144~1~1 11 Carrier, W.L. anti Seth)w, R,B. (1970) J. ~©te~ioL 1~)2,178---159 12 Nakayaraa, H., Okubo, S. and T a k ~ i , Y. (1971) ~o~1~i~. Biophys, A ~ 2 ~ , ~ - ~ 13 Braun, A. and Gros~man, L. (1974) P-~oc. Nat:, Acid. ~cL U,S, 7 1 , 1 8 ~ - - 1 ~ 4 ~ 14 Van Lancker, J,L, and Tomum, T. (1974) Bic~-~h~m.Bioph).s. A c ~ 3 5 ~ ~ 1 1 4 15 Diirwald, H. and Hoffmann-Ber]~ng, H. (1968) J. MOl. Biol. 3 4 , 3 3 1 - 0 3 ~ 16 Weiss, B., Live. T.R. and Rieha~'dson~ C.C. (1968) J. Biol. C l ~ , ~43, ~ 5 3 ~ - - 4 ~ 2 17 Seklguchl, M., Yas~da, S,, Okubo, S., Nakay~ma, H., Sblm~i~, K. ami T ~ i , Y. (19'~0) ~, ~,~I, ~ 47, 231--242 18 Alberts, B.M., Amsdio, F J,, Jetlkin~, M,, Gutman, E,D. and F~rr~, F,~, ~196~) C o ~ S ~ t1~ Symp. Quant. Biol. 33, 289--30~ 19 Lowry, O.H.~ Ros~bro~q;h, N,J,, F~rr, A.L. and R ~ a l ] , R,J. (1951) J. ]S~, C ~ . I~3, ~ 29 Sekiguchi, M. and Cohen, S,S. (1964) J. MoL Biol. 8, 638--65~ 21 Wiberg, J.S., Di~ksen, M,, Epstein, R.H., L~r~, S.E. and ~ u c 1 ~ , J , ~ , (19~:~ ~ , ~ I I , Ac~1~ $ ~ U,S, 48, 293--302 22 Nishida, Y,, Yasud~, S. and Sekiguchi, M. (1976) Binehim. Biophys. A ~ a 44~. ~8--21,~ 23 Razzell, W.E. and Kho~ana. H.G, (1961) J. BioL Ch~ra. ~36,1144--1149 24 Kushner, S.R., Kapl~n, J.C.~ Ono, H, and Grossman, L, (1971) ~ o c h ~ 10, 3325-3334 25 Kapl~., J.C., Knshner, S.R. and Grossman, L. (1971) B l o r . h ~ s ~ 10, 3 3 1 5 - - ~ 3 ~ 26 Friedherg, E.C~ (1972) Murat. Res. 15,113~123 27 Ohshima, S, and Sekiguchi, M. (1972) Btochem. Biophys, Res, C ~ , 4"~, 1 1 ~ - - 1 1 3 2 28 Shlmlzu, K. and Seklg~chi, M, (1976) J. Biol. Chem, 251, 2 6 1 3 - - ~ 1 9 29 S~to, K. and Sehi~uchl, M. (1976) J, MoL Biol, 102,15--26 30 Taketo, A., Yasud~, S. and Sekiguchl, M. (1972) J. MoL 5io], 70,1--14 31 T~naka, K., Sekiguchl, M. and Oki~la, Y. (1975) P~oe. Nail, Acad, SeL U,S, ~2, 40"~1---~?~