Function of Nucleoside Triphosphate and ... - Semantic Scholar

JOURNALOF BIOLOGICAL CHEMISTRY Vol. 256,No.15,Issue of August 10,pp. 8039-8044.1981 Prrnted in U.S.A.

THE

Function of Nucleoside Triphosphate and Polynucleotide in Escherichia coli recA Protein-directed Cleavageof Phage X Repressor* (Received for publication, January 30, 1981, and in revised form, April 23, 1981)

Nancy L. Craig$ and JeffreyW. Roberts From the Sectionof Biochemistry, Molecular and Cell Biology, Wing Hall,Cornell University, Ithaca, New York 14853

Escherichia coli recA protein catalyzes a specific pro- presence of a nucleoside triphosphate. The ATP analogue teolytic cleavage of repressors in vitro when it is acti- ATP$’ (17) substitutes for ATP in two reactions of recA vated by interaction with a single-stranded polynucle- protein: repressor cleavage (4,5,9),and thepartial unwinding otide and a nucleoside triphosphate. The ATPanalogue of duplex DNA in the presence of single-stranded DNA (18), adenosine-5’-0-(3-thiotriphosphate)(ATPyS) satisfies the latter areaction that presumably represents theinitiation the N T P requirement. We show here that despite its of DNA strand exchange. Despite being active in these reacactivity in repressor cleavage, ATPyS is hydrolyzed at tions, ATPyS is a potent inhibitor of both the complete DNA a negligible rate by the recA protein DNA-dependent strand exchange reaction (2, 18) and the recA DNA-dependnucleoside triphosphatase activity. In the presence of ence ATPase activity (4). We show here that ATPyS binds in DNA, ATPyS binds tightly to recA protein in a complex a tight complex with recA protein in the presence of singlethat can be detected because it is trapped by a nitro- stranded DNA, and is hydrolyzed only very slowlyby the cellulose filter. One ATPyS molecule is bound per recA recA triphosphatase activity. These results support our premonomer. Theseresults suggest that a ternary complex of mcA protein, DNA, and nucleoside triphosphate is sumption (4) that a ternary complex of recA protein, DNA, the species active in repressor cleavage. The activation and nucleoside triphosphate is the active species in proteolytic of recA protein by small, defined oligonucleotides in cleavage, and probably also in the reaction that initiates DNA strand exchange. We also determine that further purification place of DNA is described and characterized. of recA protein does not identify any other macromolecular components required for repressor cleavage, and we show that small oligonucleotides efficiently support h repressor cleavage.

The protein encoded by the Escherichia coli recA genehas two activities that involve a polynucleotide and a nucleoside EXPERIMENTALPROCEDURES‘ triphosphate such as ATP: itpromotes the pairing of singlekterialr stranded DNA to itshomologous sequence in a DNA duplex, DNA w a s preparedbyphenol extraction of phage p u r i f i e d bybandingtwice on C C Il grad7ents. DNA was denaturedbyheatlng a t 100’ f o r 5 minutes and q u i c k c h i l l i n g ; i t was an ATP-dependent reaction called “strand exchange” (1, 2); 1.3. O l i g o n u c l e o t i d e r assumed t h a t a solution of 50 uglml has an absorbance a t 260 m and itcatalyzes a specific proteolytic cleavage of repressors, a were Obtdlned frm P-L B l o c h m i c a l r . reaction that requires both a nucleoside triphosphate and a 14C-AlV-~-Swas a generous g i f t o f C. ATP-7-8 was obtained frm Boehringer-Mannheim. Roberts and was pPeparedby t h e method o f Goody g . (19).35S-ATP-~-S was a generous g i f t polynucleotide (3-5). recA protein also has aDNA-dependent o f Dr. F r i t zE c k s t e i n . nucleoside triphosphatase activity (5, 6) These activities reL y l o z y m was o b t a i n e d f m m Y o r t h l n g t a n B i o c h m i c a l C o r p o r d t l o n i n a l i d i x l c a c i d war obtainedfromCalbiochemIncorp.;bovlne rerun albuminandhemglobin-agarole *eve obtalned flect various roles of recA function in certain cellular processes from Sigma; u-dminohexyl-agarose was obtained frm Miles LaboPatoriel; and h y d r o x y l a p a t l t e (sometimes called SOS functions) that promote the repair of was obtained from Clarkron ChemicalCmpany. damaged DNA (7, 8). The ability of recA protein to destroy lambda repressor Prepardtlon repressors by proteolytic cleavage underlies its regulatory role s t r a l n 294(pKE277) (20) which contains reprerLambda repressor was p u r i f i e d from r. 10r a s severalpercent O f thetotalcellproteln;purlflcdtion was followed by polyacrylamide in directing the expression of genes that encode DNA repairg e lI n a l Y P I s .C e l l s were grown i n 100 l i t e r batches ~n d New B r u n r v i c kF e m t r a n fermentor related functions (3, 4, 9-11). A primary target of this protea t 37” ln medium c o n t a i n i n g 5 gm NaCl, 10 gm y e a s t e x t r a c t and 16 9” t r y p t o n e p r l i t e ? . Uhen t h e O f t h ec u l t u r er e a c h e d 0.75, t h ec e l l s were h a r v e s t e di n I Sharplercontinuous olytic activity is the E. coli lexAgene product (9), which and s t o r e d a t -20” u n t l l use. flowcentrifuge.frozen, probably acts asa repressor of both the recA gene itself(9,10, The p u r i f i c a t i o n method was m a d i f l e d f r o m s u e r and Anderegg 121). A l l s t e p s were c a w e d o u t a t 4’. C e l l s were thawed and suspended ,n 1.5ml per 9” c e l l s Lysls Buffer(0.1 M 12) and othergenes involved in DNA repair (11).recA protein T r i r HCl. pH 7.9 a t 0.2 M KCl,1 G44 EDTA, 2 vi4 CaC12, 10 mM MgC12. 5% g l y c e r o l and also directs the cleavage of the immunity repressors of tem3 G44 d l t h l o t h r e l t o l ) . A f t e r c e l l l y s i s by r o n i c a t l o n and a d d i t l o n 3 v o l u w r R Buffer [ l o perate bacteriophages such as A and P22 (4, 13), the critical G44 T r i l HCl, pH 7.9. 1 nN EDTA. 2 G44 CaCIZ, and 5% (VIVI g l y ~ e l o l ] c o n t a i n i n g a l s o 0.2 M KC1 and 3 G44 d l t h l o t h r e l t o l . c e l l d e b r i s was removed by c e n t r i f u g a t i o n f o r 45 m i n u t e l a t 5000 x event in recA-dependent prophage induction in response to g. P o l y m n P (10% w l v . adJuSted t o pH 7.9 withHCl) was added w i t h s t i r r i n g t o t h e s u p e ~ n l DNA damage. The ability of recA protein to promote the upon c e n t n f u g a t i o n (22). The t a n t t o 1.5 t i n e s t h e concentration r e q u i r e d f o r c l e a r i n g m i x t u r e was s t l r r e d 15 m n u t e s and c e n t r i f u g e d 15 m l n I t 5000 x 9. and t h e p e l l e t was suspended pairing of DNA strands reflects its direct role in homologous l n 112 t h e o n g i n d l w p e r n a t m t volume o f R Buffer conta7ning 0.2 M KC1 and 3 nH d i t h i o t h r e i t o l . recombination (14, 15), including recombinational repair of The m i x t u r e was s t i r r e d 15 minand c e n t r i f u g e d 15 rn?n a t 5000 x 9. and t h e r e s u l t i n g p e l l e t damaged DNA (16). ’ The abbreviations used are: ATP& adenosine-5’-0-(3-thiotriWe present evidence here that recA protein is activated to cleave repressors by binding to single-stranded DNA in the phosphate); NTP, nucleoside triphosphate. * Portions of this paper (including “Experimental Procedures” and * This workwas supported by Grant GM21941 and a Career Table I) are presented in miniprint as prepared by the authors. k

Of

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Development Award (to J. W. R.) from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ Present address, Laboratory of Neurochemistry, National Institute of Mental Health, Bldg. 36, Room 3D30, Bethesda, Md. 20205.

Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, Md. 20014. Request Document No. 81M-207, cite author(s), and include a check or money order for $3.60 per set of photocopies. Full size photocopies are also included in the microfiiedition of the Journal thatis available from Waverly Press.

8039

recA Protein Cleavage Activity

8040 a d d l t r a n Over 30 n i n t l o n a l 30 m l n .

15 rn?n a t 5000 x 9.

40 9 (NH4IZSO4 per100 "1 s u p e r n a t a n t , f o l l o w e d b y s t i r r i n g an addrcentrifugation f o r 45 min a t 5 w O x 9, washed

Of

40 g (NH4)2S04 per100

R buffercontaining

d

M KC1 and 3 mM d i t h i o t h r e l t o l , anddialyzedagainstthe

R Buffercontaining0.1

flowrate

115columnvolume

Of

Were a p p l i e d p e r

t h eP r o t e i n

1111

R Buffer containing

Of

e q u l l l b r d t e d i n 0.1 M KPi. pH

columnbedvolume.

Unltlof

I n 5%

0.190

2.1

0.905

10.1

3.42

7

0.596

6.6

3.21

I b l Per mol & P r o t e i n O f t h e &gene (28,291,

(11

(21 Concentration

2

frm 0.1 H KPi pH

5 rrM d l t h l o t h r e i t o l , and c l a r l f l e d b yc e n t r i f u g a t i o n .P m t e i n

addltlon

Of

42 9Wml(NH4)ZS04/100mlofsupernatant.dissolved

KC1 and 5 fl d I t h i o t h r e T t o 1 , d i a l y z e d a g a i n s t t h e The supernatant was loadedonto a 11 ml 10.30%

0.55

i n R Buffercontaining

d i l u t e dw i t h

re-

(NH4)ZS04.and

p u n f l e d by g l y c e r o l g r a d r e n t r e d m e n t a t r o n a s descrlbed above. Fldctlonscontainingrepm610p N2 u n t i l used.Repressorprepared i nt h i sf a l h l m i s g r e a t e r than 99% pule by polyacrylamlde g e le l e c t r o p h o r e s i s .

*ere f r o z e n q u l c k l y and s t o r e d i n l l q u l d

t h ee x p e r l m n to fF l g . 1, r e p r e s s o r was pdSIed over a column o f h e m g l o b l n agarore a f t e r h y d r o x y a p a t i t e Chromatography; a f t e r concentration, one c y c l e of g l y c e r o l FOP

gradlentCentvlfUgatlOn a r e removedby

Prepardtl'ln

from

average

mglrnl o i

gels,prepared 1n 01%

(wlvl

i

comparable topvotelnprepapedfrom

a (25a) ( a k i n d g i f t o f

p M11187.

m l ) was u s u a l l y p u r l f i e d

a c e t i ca c i da n d Of

andSubJeCted

The d m u n t

Of

ln

s t r a i n JAZOO (pLC 30-201 (26). C e l l s were grwn d l d e r c r l b e d above f o r r e p r e s s o r p u r i f i c a t i o n . O f t h e C u l t u r e reached 0.6, n a l l d l x l c a c l d d i s s o l v e d a t 10 mp/ml i n 0.1 M NdOH was added t o a f l n a i Concentration O f 40Lglml t o Inducethearnpliflcation Of proteln.

6.8.

0.01% (ulv)

and 201 ( v l v )

M I d e t e r n l n e db yd e n s l t o m t r i c

pulck Scan Flur-VirOenritometer,using frm a Peactlonmixture

scana 545

i n which a known amount

reprerror was completely cleaved were used.

Of

and ATP h y d r o l y s l r

ATP-1-8

were supplemented w i t h nOn-?ddioaCtlVe u7srble maPker. f a l l o w e d b y t h i n - l a y e r Chromtogra-dphy

hydrolysis,reactionmixturer d

0.75 M KPl (pH 3.5) on ~ e l l ~ l o s e p l a t eimpregnated r w t h polyethyleneimine(Brlnkman ATP-1-8

and ADP were d e t e c t e d b y v l w i n g i n i nR n n l f l u o r

ADP p r e s e n t i n l t i a l l y i n t h e p m p a l a t l o n

i.

60 mM T r i r HC1,pH

r e p r e s s o rc l e a v e d

Ar 1 s t a n d a r d , r e p r e r r o r f r d m n t s

I n r t r u m e n t r )( 3 2 ) .

was p u r i f i e d from

The

5.9.

t o e l e c t r o p h o r e s l l on 10-251gradientacrylamlde

a HelenaLabaratorles

s t a i n e dg e l sw i t h

nm f i l t e r .

radiography. u t Out.andcounted

E. P h ~ z ~ c k y )i;t was

% proteln

Wildtype

i

a s describedbyStudier(31).Gels were s t a i n e d a t 37O f o v a t l e a s t t n hours 3.5% (u/v) r u l f o r a l l c y l r c acid. 11.5% ( w l v ) t r l c h l o r o 296 ( V l v ) methanol Or e t h a n o l , and d e s t a i n e da t 37' i n 7.5% ( v l v ) a c e t i c a c l d

and 5 % ( v l u ) e t h d m l .

ning

a t 100'.

R7P-7-8 t o 4 nN t o prOYIde c a r r i e r and

procedure from c e l l s I n f e c t e d v l t h

DNA sequence.

was 3.47 r n g l n gl ,i v i n g :

2% ( w l v ) sodium dodecylsulphate

0.4 M 0-mercaptaethanol, 2 .in

k a r u r e m n t O f ATP-1-5

protein

e

absorbance 2.05 a t 280

C o a m s s i eB r i l l i a n tB l u e ,

Forassay

& p r o t e l nm o d i f l e db yt h em u t a t i o n &44l (fannerlycalled E. I t r l l n DM1187 ( 2 5 ) . Ue o c c a s i o n a l luys epdr o t e lpnr e p a r ebdt yh l l

35, t o show a g r e e w n t

&pmtein in Iolut?on of

Samples were brought t o a f i n a l C o n c e n t r i t r o n o f brnxlphenolblue. 9lYCerol.heated

hemoglobin-agarose (24). r e p ~ e ~ s owas r n o tr e t a i n e d .

Of

amino a c i d s n o m l i z e d b y s e t t i n g g l y c i n e t o DNA sequence.

a11 d e t e m i n a t l o n lse x c l u d l n gt y r )

Of

2 . 0 5 a t 280

on a Beckman 119 Amno a c i d a n a l y z e r a f t e r a c l d

P o l y a w y l a m d eg e la n a l y s i s

Of

Some p r o t e a s e s .p o s s i b l yi n c l u d i n gt r a c ec o n t a m l n a n t r ,

was performed.

DNA sequence analy611

& p m t e i ns o 1 u t l o no fa b s o r b a n c e

0.2 M

by c e n t r i f u g a t i o n . 10 rrw T r i s ,

an equal volume of R

0.2 H KC1 and 10 mH d r t h r o t h r e i t o l , c o n c e n t r a t e d w i t h

3.59

m. Using OlOleCUlar w e i g h t Of 37,773 and Cmpositlondeterminedby

( V I Vl lIn e a r g l y c e r o l g r a d i e n t i n

repressor were fiaaled,

The f r a c t l o n rc o n t a i n l n g

BufferCOntllnlng

llearuredcontentof w i t hC m p O I i t l O nb y

(41 C a l c u l a t e dC o n c e n t P a t i o ni n

0.2

PH 7.9,1 nP EDTA, 2 rrH CaClZ, 0.2 M KC1 and 10 mH d l t h i o t h r e i t o l and c e n t r i f u g e d f o r 40 h o u m a t 35,000 r p m i n a Beckman SY41 Totor.Fractions were c o l l e c t e d from t h et o p of t h e gradlent.

(31

was p r e c i p i t a t e db y

same b u f f e r , a n d c l a r i f r e d

s e l e c t e d amino a c i d r ,d e t e m > n e db y

Of

of amino a c i d s i n a

determinedbyaminoacidanalysis h y d r o l y r l r( 3 0 ) .

6.8 t o 1.0 M KPi pH 6.8

g l y c e r o l and 3 nH d i t h i o t h r e i t o l .R e p r e r r o re l u t e df r o mt h ec o 1 u ma ta b a u t Fractions c o n t a i n i n g repressor were pooled.dlalyzedagalnrt R Buffercontaining

H KC1 and

Iddl

"A.

(V/V)

M KPl.

3.43

2

yolume p e r hour t o a h y d r o x y l a p a t i t e column

l l n e a rg r a d l e n t

3.49

35.0

10

Column volume o f 0.1 M KPi,

and e l u t e d u l t h a 15-columnvolume

35.6

and r e p r e s s o r was e l u t e d

were a p p l l e dp e rm lo fc o l u m bed vcllume. The c o l u m pH 6.8, 5% ("1") g l y c e r o l and 3 nM d i t h i o t h r e i t o l ,

ONA-agarore f r a c t l o n p r o t e l n

washed w i t h 1

Ma6

3.23

The c o l u m was washed w i t h 2 column

5% (VI") g l y c e r o l and 3 *I d i t h l o t h r e i t o l ; a b o u t

6.8.

3.45

35

3.18

0.1 M KC1 and 5 d4 d i t h i o t h r e i t o l .

a flowPateofabout1column

3.49

14.1

35

d i t h i o t h r e i t o l and 5% ("1") glycepol.andclarifiedbycentrifugation.Thissupernatant

was a p p l l e d a t

38.6

u n i t so f

40

With R B u f f e r c o n t a i n i n g 0 . 4 M KC1 and 5 n*l d l t h l o t h r e r t o l . FPaCtions c o n t a i n i n gr e p r e s s o r were pooled,dlalyzedagainst10 d4 T r i s H C l . pH 7.9,0.05 "#4 EDTA, 2 mH CaC12, 0.4 M KC1. 5

rrM

38 14

a rlnglestranded

0.1 M KC1 and 5 mM d i t h i o t h r e i t o l a; b o u t

Of

3.51 1.28

5000 x 9. thesupernatant was app17ed DNA agarore column (23).

p e rh o u rt o

e q u l l l b r a t e d In R B u f f e cr o n t a i m n g volumes

0.2 M KCl, 3 n*l d i t h i o t h r e i t o l and

45 min a t 5000 x g , d i s m l v e d i n

1111 f o l l o w e db yc e n t r i f u g a t i o nf o r

sane b u f f e r . A f t e r c l a r i f l c a t i o n b y c e n t n f u g a t l o n a t

at

ThlS

P r o t e l n was p r e c i p i t a t e db y

A p r e c r p i t a t e was c o l l e c t e d b y

tW1Ce by I t l r r l n g 10 min i n 1/2volume 1/40volume

0.6 M KC1 and 3 "#4 d i t h i o t h r e i t o l .

R b u f f e rc o n t a l n l n g

suspended I n 112volumeof

V11

m i x t u r e was r t l r r e d 15 minandcentrifuged

Of

ultraviolet l l g h t or byauto-

(New EnglandNuclear).COrreCtlOn

ATP-1-8,

vas made f o r

u s u a l l y 15-20% theConcentrationofATP-i-S.

ATP hydrolysis YIP measured as d e s c r i b e d (4).

When t h e

C e l lg m w t h

"dl

f l o wr e n t r l f u g e .

c a n t l n u e df o r 45 m l n and t h e c e l l s were h w v e s t e d l n a SharplesContlnUOUI The c e l l p e l l e t was resuspended i n 3.3 ml 50 m TP MIS HCI, pH 7.5 and 10%

( w l v ) sucrorelgm o f c e l l s , f r o z e n ,

-20-CU n t l l use.

and s t o r e d a t

The p u r l f l c l t i o np r o c e d u r e

was a m o d l f l c a t i o n

t h e method d e r c r l b e d (3.4.5).

Of

Purity

The c e l l l y s l l procedure was m d l f l e d from cell rurpenrlan was thawedand distributed t o Beckman 30 0.025 "01 O f 4 mgfml lysozyme and 0.025 "01 o f 4 M NaCl were added. The s l x t u r e was kept on ~ c 30 e mln, incubated a t 3 7 O f o r 6-8 m i " , c h l l l e , and c e n t r l f u g e df o r 45 oln a t was monitored by polyacrylamide g e la n a l y s l r .

Schekman e t a1. ( 2 7 ) .A f t e rt h e r o t o rt u b e s ,

20.000 rpm i n t h e Beckman 30 mtw. r u l f a x l d e and0.0025volumes NaCl was added t o 0 . 2 M .

l h eI u p e r n a t a n t

was m x e d w t h 0.0075 v o l o f d i m e t h y l -

o f 0.2 M pbenylmethylrulfonylfluarlde 10% ( v f v ) Polymln P ( a d j u s t e dt o

~n d l m e t h y l r u l f o x l d e ,

and

pH 7.9 w t h HCl) *as added m t h

r t l r r l n q t o a f l n a l concentration 1 . 3 t i m s t h e m w n t r e q u l r e d f o p c l e d r l n g ( 2 2 ) . A f t etrh e m l x t u r e was s t l r r e d 15 m l n and c e n t r r f u g e d 15 mln a t 5000 x g . t h e p e l l e t was resuspended

o f X Buffer [20 mH Trll-HC1. pH 7.5. 0.5 Rw EDTA, 1 rd4 C o n t d i n l n g0 . 5 M NdCl. The m i x t u r e was s t i r r e d 15 m7n a t 5,000 x g. The p e l l e t was suspended In 1 v o l u m X BufferCOntdlnlng

w t h one orlglnalsupernatantvolume d l t h i o t h r e l t o l and 10% ( w f v ) a n dc e n t n f u g e d

15mi"

SUCTOIB]

1 0 M NaCl. i t l l r e d 15 mln and c e n t r i f u g e df o r

15min

over 30 m i n o f 28

frm thesupernatantbyadditlan

a t 5.000 x 9.

P r o t e l n was p r e c l p l t a t e d

gm (NH4)2S04 per100

m l ofSUPerndtdnt,

A p r e c l p l t l t e was c o l l e c t e db y c e n t r i f u g i n g 4 5 m ~ an t 6.000 x g . washed by s t i r r i n g 15 mln ~n 1 volume X B u f f e r c o n t a i n i n g 0.15 M NaCl and 0 . 2 8 grn (NH4)ZS041 m1. and c o l l e c t e db yc e n t r l f u g a t l o n . The p e l l e t was e x t r a c t e d by r t l r r l n g followedby

r t l r r l n g an a d d i t l o n a l 30 mi".

20 mi" i n 112 volume X Buffercontainlng

0.15 H NaCl and 0 . 2 0 gm (NH4IZ2O4 per m l , followed

35 m n a t 6.000 x g .

b yc e n t r l f u g a t l o nf o r

P r o t e l n was p r e c l p 7 t a t e d from t h eP e l u l t l n g

by CentPlfUgatlOn, Supernatant by the d d d l t l o n Of 0.15 gm (NH4)ZS04/rnl O f s u p e r n a t a n t .c o l l e c t e d dissolved I " 1/40 volume P B u f f e r 120 "#4 KPi. pH 6.5, 10% ( v f v l glyCePDl. 0.1 mM EDTA and 1 nM d l t h i o t h r e l t o l ) , and d l a l y z e da g a l n r tPb u f f e r .A f t e rc l a r i f i c a t i o n by C e n t r i f u g a t l a n I t 5000 x g , t h e s o l u t i o n

was a p p l l e d a t

a flowrate

p h o c e l l v l o r e column e q u i l i b r a t e d ~n P Buffer;

o f 114columnvolumePerhour

a k u t 1 A''

u n l to fp r a t e l n

t o a PhoSwas a p p l l e d Per

1.5 rnl o f res?". The column was washed w i t h 1.5 col~lllnvalumer Of P B u f f e r and p m t e l n was e l u t e d w l t h a n 8-column v(11ume lrnesr gradlentfrom zero t o 0.25 M KC1 i n P Buffer. ECJ p r o t e i n e l u t e d frm t h e column a t a b o u t 200 mM KC1. f r a c t i o n sc o n t a i n l n g & p r o t e l n were pooled.dlalyzed Buffercontainlng

against Pbuffer,adsorbed

Of

and e l u t e d W i t h

agaln t op h a r p h o c e l l u i o r e .

Fractions c o n t a l m n g

0.25 M KC1.

p r e c i p i t a t e db yt h ea d d l t i o n

& p r a t e l n were

P

ponled and protein

Wdl

0.4 gn (NH4)2S04/ml o fr a l u t l o n ,f o l l o w e db yC e n t r l f U g a t l o n .

The p e l l e t was a l r r o l v e d ~n 20 lrEl T r i s HCl, pH 7.5,0.5

nd4 EDTA. 1 mM d i t h i o t h r e l t o l .

0.2 M

ram b u f f e r .D e b r l s * d l removed by cenwax loaded Onto an 11 ml 6.20% (WIVI l i n e a r IucrOSe g r a d l e n t

NaCl and 2% ( w / v ) sucrose and d i a l y z e da g a i n r tt h e t r i f u g a t l o n and thesupernatant i n 20

rrw

T r i r HCl, pH 7.5, 0.5 n*l EDTA. 1 nM d i t h l o t h r e i t o l ,

and 0 . 2 M NaCl.

A f t e rt h e

g r a d l e n t was c e n t r l f u g e d f o r 36 h o u r s a t 38,000 rpm i n a Beckman SW41 rotor. f r l C t l O n l were c o l l e c t e d flm thetapofthegradient.Fortheexperiment O f F l g . 1, t h ep U r i f i C a t l O n Y.33 extended a s f o l l a r . ChromltOgTaphy. 0.10

n

A gradientelutlm

FractlOn6 contalnlng

NaCl andpassed

p r o t e i n was n o tr e t a i n e d . I"

was used f o r t h e

& p r o t e l n were

over a columnofhemglobin-agarore

The p r o t e i n t h e n

second c y c l e of p h o r p h o c e l l u l o r e d l a l y z e da g a i n s t (24)

I"

X b u f f e r containing

t h e same b u f f e r , AE

was adsorbed t o a columnof

t h e same buffer- and e l u t e d S t e p w i s e with X b u f f e r c o n t d l n i n g

u - m l n o hexyl-agarose 0.50 M NaCl. A f t e r COnCentPd-

t i o n , t h e p m t e i n was redimented on a ~ u c r o s eg r a d l e n t a s described above. The concentPation o f protein was c a l c u l a t e d f r o m t h e measured extinction c a e f f l c i e n t , = 5.9 ( T a b l e I ) . The r a t i o A2Bo/A260 Of p u r l f l e d & p r o t e i n was 1.59.

e

RESULTS

X Repressor Cleavage Activity Is Associated with Extensively Purified recA Protein-Incubation of purified A repressor with purified recA protein, ATP, and polynucleotide results in the specificproteolyticcleavageof the repressor polypeptide (3, 4). recA protein is required for this cleavage reaction, because the cleavage activity co-purifies with recA protein, and mutations in the recA gene alter the repressor cleavage activity of purified recA protein in vitro (3, 33, 34). Since it is surprisingthat recA protein should bea protease in addition to its other activities, we extended our purificationto several steps beyond the stage of apparent homogeneity for both recA protein and X repressor, to lessen the probability that a separate unidentified trace component is required for repressor cleavage. Fig. 1 shows that cleavage activity still copurifies with the recA polypeptide in the final stage of this extensive purification,sucrosegradient sedimentation. The specific activity of cleavage is uniform acrossthis gradient and is identical with that of the precedingseveralpurification steps. Thus we find no evidence that any proteinexcept recA is required for cleavage, and we presume that the recA polypeptide contains the catalytic site for proteolysis. Since recA protein clearly is at least essential to the reaction, noneof our conclusions in this paper (or elsewhere) would be affected if an unidentified minor component wereinvolved. ATP@ Is Hydrolyzed Very Slowly by recA Protein-In the presence of polynucleotide, recA protein catalyzes the hydrolysis of ATP to ADP and Pi (5, 6 ) . The ATP analogue ATP+, which both promotes repressor cleavage andinhibits the ATPase activity of recA protein at micromolar concentraThe tions, is itself hydrolyzed very slowly by recA protein. products,identifiedby polyethyleneimine chromatography andautoradiography, are ADP (from [14C]ATPyS) and a species we presume to be thiophosphate (from [35S]ATPyS).

Activity Cleavage Protein recA

804 1

Like repressor cleavage and ATPase activity, ATPyS hydrolysis activityco-sedimentswith recA proteinon asucrose I1 shows that at 2.3 PM gradient (data not shown). Table ATPyS, the rateof hydrolysis is about '/so00 of that of ATP at 5 mM. Since 2.3 PM is near or above the K , for ATPyS hydrolysis inthese conditions (data not shown),hydrolysis the rate is negligible at any ATPyS concentration. The turnover number for ATPyS hydrolysis is one per several hours, versus one per several seconds for ATP. This result suggests strongly that a complex of recA protein with ATPyScleaves repressor, and that hydrolysis of thenucleotide is notrequired for cleavage activity. If hydrolysis of ATPyS were required for repressor cleavage,

A

A A

FIG. 2. The rate of repressor cleavage is independent of the extent of ATPyS hydrolysis. Reaction mixtures containing 11 mM

I

,

20 BOTTOM

I 18

,

I 16

,

I 14

I

I 12

Fractlon

TOP

FIG. 1. Repressorcleavageactivity co-sediments with highly purified recA protein. Reaction mixturesof 40 pl contained 12 mM Tris-HCI, pH 7.5, 1mM potassium phosphate, 0.55 lll~EDTA, 0.4 mM CaC12, 2.4 mM dithiothreitol, 0.045 M NaC1, 0.015 M KC1, 5% (w/v) sucrose, 4% (v/v) glycerol, 1 mM ATPyS, 2 mM MgCL, 10 pg/ ml 4 pgof repressor, and recA+protein as indicated. Fractions of equal size were taken across the peak of a sucrose gradient of recA protein, the last step in the extended purification described under "Experimental Procedures." The purification of X repressor included chromatography on hemoglobin agarose. After incubation for 105min at 37 "C, reactionmixtures were analyzed by polyacrylamide gel electrophoresis. 0, micrograms of recA protein per reaction; 0, micrograms of X repressor cleaved; A, nanograms of repressor cleaved/ min/pg of recA protein.

TABLEI1 Rates of ATP and ATPyS hydrolysis Reaction mixtures of 25 pl contained 10 mM Tris-HC1, pH 7.5,0.25 mM EDTA, 0.5 mM dithiothreitol, 5% (w/v) sucrose, 0.06 M NaC1, and 20 p g / d of heat-denatured X DNA. Reaction mixtures in which ATP hydrolysis was measured contained in addition 10 mM MgCL, 5 mM [y3'P]ATP, and 1pg of recA+ protein; they were incubated 60 min at 37 "C, followed by chilling on ice to stop the reaction. Reaction mixtures in which ATPyS hydrolysis was measured contained, in addition, 3.3 mMMgC12, 2.3 PM [I4C]ATPy-S and 0.86 pgofrecA' protein, and were incubated 45 min at 37 "C, followed by chilling on ice. NTP

ATP ATPvS

Rate nmol/min/mg recA protein

350 0.067

Tris-HCI, pH 7.5, 0.4 mM EDTA, 1.6 mM dithiothreitol, 6% (w/v) sucrose, 1 mM Tris-HCI, pH 7.9, 2% (v/v) glycerol, 0.2 mM CaCL, 3.7 p~ [14C]ATPyS,2.6 mMMgC12, 1 mM potassium phosphate, pH 7.0, 0.02 M NaC1,0.02 M KCI, 3.3 pg/ml of denatured X DNA, and 113 pg/ ml of recA441 protein were incubated at 37 "C for 5,10,30, or 45 min. One-tenth of a volume of 10 mM Tris, pH 7.9, 20% (v/v) glycerol, 1 mM EDTA, 10 mM dithiothreitol, 2 mM CaC12,0.2 M KCI, and 1.5 mg/ ml of h repressor was added, and incubation was continued at 37 "C. At 5-min intervals, aliquots were removed and chilled, and ATPyS hydrolysis and repressor cleavage were assayed, from 25- and 50-pl portions, respectively. A, time course of repressor cleavage after different times of preincubation. The rate was determined by the slope of the line in the interval 5-15 min (except at 45 min when the interval 5-10 min wasused). Because the cleavage reaction is inhibited by high salt (4), the decrease in rate of cleavage after the first interval could result from a slow response to theincrease in salt concentration upon addition of repressor. B, extent of ATPyS hydrolysis and the rate of repressor cleavage. The ATPyS hydrolysis represents incubation both before and after addition of repressor, but repressor did not affect the rateof ATPyS hydrolysls. The rateof repressor cleavage is plotted against the time at which repressor was added.

it is possible that some species resulting from thishydrolysis would accumulate during incubation, causing the rate of repressor cleavage to increase continuously. In the experiment of Fig. 2, we examined ATPyS hydrolysis and repressor cleavage in the same reaction, by incubating recA protein and ATPyS together in conditions of the cleavage reaction and removing samples at intervals to measure both their ability to cleave repressor and the extent of ATPyS hydrolysis. Fig. 2 shows that the rate of repressor cleavage is independent of the time of preincubation of recA protein with ATPyS and with the extent of ATPyS hydrolysis. This experiment also shows that stoichiometrichydrolysis of ATPyS is not required for repressor cleavage: in the 15 min following addition of repressorafterthe 5-min preincubation, 8 X lo-" mol of repressor were cleaved while 0.6 X 10"' mol of ATPyS were hydrolyzed per 50-pl sample, more than 10 cleavage events

bound

recA Protein Cleavage Activity

8042

per molecule of ATPyS hydrolyzed. We conclude that if any species dependent upon ATPyS hydrolysis is required for repressor cleavage, it exists in very small concentration and does not accumulate during the reaction. Since ATPyS promotes repressor cleavage more efficiently than do nucleoside triphosphates that are hydrolyzed much faster (34), it seems most likely that a complex of recA protein with the triphosphate is the active species in repressor cleavage, and that hydrolysis of the triphosphate is not required. ATPyS Binds Tightly to recA Protein in the Presence of DNA-The finding that ATPyS at micromolar concentrations efficiently stimulates recA protein to cleave repressors, even though it is hydrolyzed extremely slowly, suggeststhat ATPyS forms a relatively stable complex with recA protein. The experiment of Table I11 shows that radioactive ATPyS incubated with recA protein in the presence of DNA is retained by a nitrocellulose fiiter. Neither ATPyS itself nor ATPyS incubated with recA protein in the absence of DNA is bound in these conditions, although recA protein alone is retained by nitrocellulose (data not shown). Thus, incorporation of ATPyS intothis complex requires both recA protein and polynucleotide. The DNA concentration dependence of ATPyS binding is similar to that of ATPase activity, in that bound ATP@ reaches a plateau at higher DNA concentrations (data not shown), and it is unlike the DNA concentration response of repressor cleavage, which is inhibited by higher than optimal DNA concentrations (see Ref. 4 and below). The binding of ATPyS is apparently irreversible, since 1)the halflife of the complex is of the order of hours, similar to the turnover number for ATPyS hydrolysis; and 2) bound radioTABLE I11 DNA-dependent binding of ATPyS to recA protein Reaction mixtures of 60 p1 contained 10 mM Tris-HC1, pH 7.5,0.25 mM EDTA, 0.5 mM dithiothreitol, 5% (w/v) sucrose, 0.06 M NaCI, 200 pg/ml of bovine serum albumin, 5.3 p~ [I4C]ATPyS,2.3 mM MgC12, 97 pg/ml of recA441 protein (2.6 p ~ (unless ) omitted), and 20 pg/ml of heat-denatured X DNA (unless omitted). After incubation at 37 "C for 20 min, 50-pl aliquots were fitered onto a quarter piece of a Schleicher & Schuell filter (BA85, 25 mm), washed with two 50-pl portions of 10 mM Tris-HC1, pH 7.5, 0.25 mM EDTA, 0.5 mM dithiothreitol. 5% (w/v) sucrose. 0.06 M NaC1. and 2mM MzCb. and counted. ATPvS

Additions pmol

None Heat-denatured h DNA recA protein recA orotein and heat-denatured h DNA

nml

mcA

0.5 1.0 2.5 115

Monomers

FIG. 3. Stoichiometry of ATPyS binding to recA protein. The assay was performed as described in the legend to Table 111, except with 5.3 p~ ['4C]ATPyS and recA441 protein as indicated. The amounts are calculated for 1 ml of binding reaction.

I 20

I 10

Free ATP-I-S,

I 30

pM

FIG. 4. Binding of ATPyS to recA protein in thepresence of single-stranded DNA or (dA)le. The assay was performed as described in the legend to Table111, except that bovine serum albumin was omitted; the concentration of ["SIATPyS wasvaried as indicated; the concentration of recA4f1 protein was 85 pg/ml; either 20 pg/ml of heat-denatured h DNA or 10 pg/ml of ( d A ) 1 6 was present; and incubation was for 5 min. Because a significant fraction of the ATPyS is bound at low concentration, the unbound concentration is plotted. The ordinate is the ratio of moles of ATPyS bound to moles of recA protein in the filtered sample. 0,heat-denatured X DNA 0, (dA),R.

FIG. 5. Single-strandedDNA dosage curve of recA proteindependent cleavage of X repressor. Reaction mixtures of20 p1 were constructed as described in the legend to Fig. 1, except with 148 pg/ml of repressor, 51 pg/ml of recA441 protein, 0.020 M NaC1,0.040 M KCI, and heat-denatured h DNA as indicated. After incubation of 45 min at 37 "C, cleavage was measured as described.

active ATPyS is not exchanged for nonradioactive ATPyS added after the complex is f ~ r m e d We . ~ presume that the trappable species is a ternary complex of recA protein, polynucleotide, and ATPyS, although we have not measured bound DNA directly. Others have shown that DNA binds tightly to recA protein in the presence of ATPyS (2, 35). There is evidence of three sorts that ATPyS is bound without hydrolysis or other covalent modification. 1) Direct examination by polyethyleneimine chromatography of material retained by the fiiter showed that more than 90%of the radioactivity was in ATPyS (data not shown). 2) We detect trapping of either ring-labeled ['*C]ATPyS orthiophosphatelabeled [35S]ATPyS.3) ATPyS hydrolysis is so slow that little could occur during the time in which complex formation is assayed. The Stoichiometry of ATPyS Binding-The binding of ATPyS to recA protein saturates at high concentrations of ATPyS, as shown below (Fig. 4). To determine the stoichi-

' C. Roberts and J. W. Roberts, unpublished results.

ActivityCleavageProtein

8043

recA

ometry of boundATPySand recA protein, we measured ATPyS bound withincreasing concentrations of recA protein, using a saturating concentration of ATPyS (Fig. 3). The slope of this curve gives 0.9 molecules of ATPyS bound per recA protein monomer. It is a reasonable presumption that each monomer has one binding site for ATPyS(andforother NTP's), although this measurement obviously is consistent with some different configuration such as two ATP@ molecules bound to half of the recA monomers. Both DNA andoligonucleotides such as (dA)16support the binding of ATP@ torecA protein (Fig. 4). The concentration la

1

1 1

I

I

I

50

H

i

v

1

s y

"

5

20

10 15 Oligonucleottde.pg/mI

1

I

I

I

I

of ATPyS bound a t saturation in the presence of this oligonucleotide is identical with that with single-stranded DNA,so that the stoichiometry is an intrinsic property of the recA polypeptide. However, a higher concentration of ATPyS is required to saturate thebinding supported by (dA)lG,suggesting that the oligonucleotide interacts with recA protein less efficiently than does DNA. Stimulation of Proteolytic Activity of recA Protein by Oligonucleotides-The proteolytic activity of recA protein toward repressors, like its ATPase and ATPyS-binding activities, requires polynucleotide. Both long denatured DNA mololigonucleotides ecules, such as phage A DNA,andsmall satisfy the requirement (4). In reactions using ATPyS, the maximum rateof repressor cleavage occurs at a concentration recA protein concentration, of DNA thatis proportional to the suggesting that a stoichiometric complex between these components is the activecomplex in repressor cleavage (4). Higher than optimal concentrations of single-stranded h DNA inhibit cleavage, as shown in the experiment of Fig. 5. We have found two differences between the activityof DNA and the activity of a series of deoxyadenosine oligonucleotides. 1) Small oligonucleotides do not inhibit repressor cleavage at high concentration. 2) The smaller oligonucleotides promote cleavage only at much higher concentrations than are required of DNA, suggesting that they do not interact stoichiometrically with recA proteininthese conditions. Fig. 6 shows that (dA)9, (dA)16, and (dA)19-24are similar to h DNA inpromoting repressor cleavage a t low concentrations, and thus presumably alsobindefficiently to recA protein. In contrast, ahigher concentration of (dA)* is required to saturate the cleavage rate, and (dA), showsonlyslight activity at much higher concentration.The oligonucleotide (dT)g is also active at somewhat higher concentrations than single-stranded DNA. Although (dA)19-24 inhibits cleavage at higher concentrations, (dA)16 andsmaller oligonucleotides do not. This result shows that the inhibitionof cleavage by high concentrations thus probably of DNA is separable from the stimulation, isand an interaction distinct from that which stimulates repressor cleavage. If recA protein is preincubated with a saturating concentration of (dA),6 in thepresence of ATPyS, and excess single-strandedDNAisthenadded,repressor cleavage is inhibited (data not shown); thus, the site or sites responsible for inhibition are notirreversibly bound by excess oligonucleotide. DISCUSSION

I 20

I 40

I

I I

60

Oltgonucleottde, pg/ml

FIG. 6. Oligonucleotide dosage cuwes of recA protein-dependent repressor cleavage. Reaction mixtures were constructed as described in the legend to Fig. 1, except with 51 pg/ml of recA441 protein, 0.015 M NaCI, 0.045 M KCI, and repressorand oligonucleotide as indicated. The maximum cleavage rates varied by no more than a factor of two among oligonucleotides; activity is plotted as a percentage of the maximum. Incubation was at 37 "C.a, (dA)lg.as;h repressor at 149 pg/ml; incubation for 51 min. b, ( d A ) I ~X; repressor at 149 pg/ ml; incubation for 51min. c, (dA)9;h repressor at 176 pg/ml; incubation for 120 min. d, (dA)s;X repressor at 176 pg/ml; incubation for 45 min. e, (dA),;h repressor at 159 pg/ml; incubation for 109 min. No saturation of the cleavage reaction was observed, and activity is plotted arbitrarily as a percentage of the maximum activity obtained with (dA),S. (dT)p;h repressor at 293 p g / m l ; incubation for 60 min.

We have shown that recA protein binds ATPyS tightly in the presence of DNA, probably in a ternary complex of all three components. Presumably, an equivalent complex forms withnaturaltriphosphatessuchasATP before theyare hydrolyzed. We have inferred that binding of recA protein intothisinitial complex, without hydrolysis of the NTP, invokes itsproteolyticactivitytoward repressors. ATPyS stimulates recA protein to cleave repressors more efficiently than does ATP (4,34), yet the rate of ATPyS hydrolysis by recA protein is several thousand-fold less than the rate of ATP hydrolysis. Furthermore, less than one ATPySmolecule is hydrolyzed for each repressor monomercleaved, indicating that ATPyS hydrolysis is not directly coupled to repressor cleavage. ATPyS also stimulates recA protein to bind and unwind duplex DNA (18),a reaction that may represent the initiation of DNA strand exchange; we presume that ATPyS (or NTP)hydrolysis alsois notessential to thisinitial reaction. Since ATPyS strongly inhibits both ATPhydrolysis and the completion of strand exchangecatalyzed by recA protein, ATP hydrolysis probably is required for subsequent steps in strand exchange that involve the alignment of homologous

recA Protein Cleavage Activity

8044

sequences during strandpairing. (1979) Proc. Natl. Acad. Sci. U. S. A . 76, 1630-1642 2. McEntee, K., Weinstock, G., and Lehman, I. (1979) Proc. Natl. Besides their response to ATPyS, a second property comAcad. Sci. U. S. A . 76,2615-2619 mon to the repressorcleavage reaction and theunwinding of 3. Roberts, J., Roberts, C., and Craig, N. (1978) Proc. Natl. Acad. duplex DNA by recA protein is that oligonucleotides satisfy Sci. U. S. A . 75,4714-4718 the polynucleotide requirement for both (4, 18).The affinity 4. Craig, N., and Roberts, J . (1980) Nature 283, 26-30 of recA protein forlonger oligonucleotides is apparently high, 5. Roberts, J., Roberts, C., Craig, N., and Phizicky, E. (1978) Cold because the longer deoxyadenosine oligonucleotides and sinSpring Harbor Symp. Quant.Biol. 43,917-920 6. Ogawa, T., Wabiko, H., Tsurimoto, T., Horri, T., Masukata, H., gle-stranded h DNA are activeat similar low concentrations. and Ogawa, H. (1978) Cold Spring Harbor Symp. Quant. Biol. The activity of oligonucleotides suggests that these reactions 43,909-915 may not require the extended polymerization of recA protein 7. Radman, M. (1974) in Molecular and Environmental Aspects of that occurs ona long polynucleotide strand (36). Mutagenesis (Prakash, L., Sherman, F., Miller, M., Lawrence, Excess single-stranded DNA inhibits bothrepressor cleavC., and Tabor,H., eds) pp. 128-142, Charles C Thomas, Springage andDNAstrand exchange (4, l), suggesting that the field, Ill. 8. Witkin, E. (1976) Bacteriol. Reu. 40,869-907 target for inhibition is recA protein, not h repressor.One 9. Little, J., Edmiston, S., Pacelli, L., and Mount, D. (1980) Proc. explanation of this inhibition follows from the observation Natl. Acad. Sci. U. S. A . 77,3225-3229 that binding of recA protein to(limiting) single-stranded DNA 10. Brent, R., and Ptashne, M. (1979) Proc. Natl. Acad. Sci. U. S. A. in the presence of NTP activates it to melt duplex DNA: 77, 1932-1936 excess single-strandedDNA could inhibitbothstrand ex- 11. Kenyon, C., and Walker, G. (1980) Proc. Natl. Acad.Sci. U. S. A. change andcleavage by occupying secondary sitesthat would 77,2819-2823 otherwise bind duplex DNA. Cleavage could be inhibited in 12. Little, J., and Harper, J. (1979) Proc. Natl. Acad. Sci. U. S. A. 76,6147-6151 several ways: because a recA protein-DNA network impenetrable by repressor is formed, or thecleavage site is directly 13. Phizicky, E., and Roberts, J . (1980) J. Mol. Biol. 139,319-328 14. Clark, A. (1973) Annu. Reu. Genet. 7, 67-86 obscured, or a structural change is induced in the enzyme. 15. Kobayashi, I., and Ikeda, H. (1978) Mol. Gen. Genet. 166,25-29 Smaller oligonucleotides might not inhibitat high concentra- 16. Rupp, W., and Howard-Flanders, P.(1968) J. Mol. Biol. 31,291tionseitherbecausethey bind too weakly to occupy the 304 17. Goody, R., and Eckstein, F. (1971) J. Am. Chem. SOC.93, 6252secondary sites, or because they cannot form an extended 6257 network of recA protein and DNA. 18. Cunningham,R., Shibata,T.,DasGupta, C., and Radding, C. Because an excess of smalleroligonucleotides doesnot (1979) Nature 281, 191-195 inhibit repressor cleavage, the rate of repressor cleavage is 19. Goody, R., Eckstein F., and Schirmer, R. (1972) Biochim. Bioproportional to the concentration of recA protein in these phys. Acta 276,155-161 conditions. Thus it is convenient to use oligonucleotides to 20. Backman, K., and Ptashne, M. (1978) Cell 13,65-72 assay the cleavage activity of recA protein, as we have done 21. Sauer, R., and Anderegg, P. (1978) Biochemistry 17, 1092-1100 22. Burgess, R., and Jendrisak, J. (1975) Biochemistry 14,4634-4638 in the experiment of Fig. 1. Activation of recA proteinto cleave repressors in vivo 23. Schaller, H., Nusslein, C., Bonhoeffer, F., Kurz, C., and Neitzschmann, I. (1973) Eur. J . Biochem. 26,471-481 occurs when cellular DNA is damaged or its replication is 24. Nayakama, T., Munoz, L., and Doi, R. (1977) Anal. Biochem. 78, interrupted, for example by ultraviolet irradiation. We have 165-170 suggested that one pathway of activation is the binding of 25. Mount, D. (1977) Proc. Natl. Acad. Sci. U. S.A . 74, 300-304 25a.Mount, D. (1979) Virology 98,484-488 recA protein to single-stranded DNA in gaps that result from these treatments (4). Any single-stranded polynucleotide ap- 26. Clarke, L., and Carbon, J. (1976) Cell 9,91-99 pears to promote repressor cleavage in vitro, even though the 27. Schekman, R., Wickner, W., Westergaard, O., Brutlag, D., Bertsch, L., and Kornberg, A. (1972) Proc. Natl. Acad. Sci. U. different activities of (dT)9 and the dA series suggest that S. A . 69, 2691-2695 there is some effect of base composition. We interpret this 28. Horii, T., Ogawa, T., and Ogawa, H. (1980) Proc. Natl. Acad.Sci. generalized activity of polynucleotide to represent the ability U. S. A . 77,313-317 of recA protein to interact with any sequence of exposed 29. Sancar, A,, Stachelek, C., Konigsberg, W., and Rupp, W. (1980) Proc. Natl. Acad. Sci. U. S. A. 77,2611-2615 single-stranded chromosomal DNA, simultaneouslyinvoking D., Stein, W., and Moore, S . (1958) Anal. Chem. 30, repressor cleavage activity and engaging this DNA in strand 30. Spackman, 1190-1206 exchange. The finding that smalloligonucleotides alsoactivate 31. Studier, W. (1973) J. Mol. Biol. 79,237-248 repressor cleavage is consistent with suggestions that degra- 32. Maxam, A,, and Gilbert, W. (1980) Methods Enzymol. 65, 499dation fragments of damaged DNA (37) also could provide a 559 33. Roberts, J., and Roberts, C . (1981) Nature 290,422-424 pathway of recA activation. Acknowledgments-We thankE. Phizicky and C. Roberts for advice and help, Dr. Fritz Eckstein for a generous gift of "S-labeled ATPyS, and Dr. R. McCarty for reading the manuscript. REFERENCES 1. Shibata,T.,DasGupta,

C., Cunningham, R., andRadding, C .

34. Phizicky, E., and Roberts, J . (1981) Cell 25,259-267 35. Shibata, T., Cunningham,R., DasGupta, C., and Radding, C. (1979) Proc. Nut. Acad. Sci. U. S. A . 76, 5100-5104 36. West, S., Cassuto, E., Mursalim, J., and Howard-Flanders, P. (1980) Proc. Natl. Acad. Sci. U. S. A . 77,2569-2573 37. Oishi, M., and Smith, C. (1978) Proc. Natl. Acad. Sci. U. S. A . 75, 3569-3573