Mol Gen Genet (1990) 224:129 135

© Springer-Verlag 1990

Use of recA803, a partial suppressor of recF, to analyze the effects of the mutant Ssb (single-stranded DNA-binding) proteins in vivo and in vitro Murty V.V.S. Madiraju and Alvin J. Clark Department of Molecular and Cell Biology, c/o Stanley/Donner ASU, Room 229 Stanley Hall, University of California, Berkeley, CA 94720, USA Received April 20, 1990

Summary. We examined the possibility that the ssb-1 and s s b - l l 3 mutants exert some of their effects by interfering with the normal function of wild-type RecF protein. Consistent with this possibility, we found that recA803, which partially suppresses recF mutations, also partially suppresses both ssb mutations, as detected by an increase in UV resistance. No evidence was obtained for suppression of the defect in lexA regulon inducibility caused by the ssb mutations. Consequently we suggest that suppression occurs by increasing recombinational repair. In vitro tests of Ssb mutant and wild-type proteins revealed that the single-stranded D N A dependent ATPase activity of RecA protein is more susceptible to inhibition than the joint-molecule-forming activity. All three Ssb proteins inhibit the ATPase activity of RecA wild-type protein almost completely while under similar conditions they inhibit the j oint-molecule-forming activity only slightly. Both activities of RecA803 protein were found to be less inhibited by the three Ssb proteins than those of RecA wild-type protein. This is consistent with the suppressing ability of recA803. We found no evidence to contradict the previously proposed hypothesis that ssb-1 affects recombinational repair by acting as a weaker form of Ssb protein. We found, however, only very weak evidence that Ssb-113 protein interferes directly with recombinational repair so that the possibility that it interferes with a normal function of RecF protein must remain open.

Key words: recA803 - ssb mutation - r e c F - Recombinational repair - Escherichia coli

Introduction The temperature-sensitive lethal mutations ssb-1 and s s b - l l 3 show similar pleiotropic phenotypes at permissive temperatures although the effects of ssb-! are less severe than those of s s b - l l 3 (Chase and Williams 1986). For example, both inhibit induction of the Escherichia Offprint requests to: M. Madiraju

coli lexA and phage lambda cI regulons, which together constitute the SOS response, and both reduce UV resistance. Until recently it was assumed that the effects at permissive temperatures reflect a role of Ssb protein in the processes of SOS regulation and repair of UV damage (Chase and Williams 1986). Moreau (1987, 1988) has, however, shown that cells overproducing wild-type (wt) Ssb protein mimic in vivo the effects of a recF mutation. Since recF mutants have a pleiotropic effect at least partially similar to that of the ssb mutants (see Blanar et al. 1984 for a summary of the recF mutant phenotype), Moreau's work raises the alternative possibility, that one or both mutant Ssb proteins interfere with normal functioning of RecF protein. Two types of tests of these alternatives are available: (1) a suppressor test in vivo and (2) an enzymatic test in vitro. To perform the first test, a previously characterized partial suppressor of recF mutations can be used. This suppressor mutation, recA803 (Volkert and Hartke 1984; Madiraju et al. 1988), alters RecA protein so that it can hypothetically dispense with the functions of RecF protein in recombinational repair of UV damage (Madiraju et al. 1988). If either or both ssb mutations interfere with such functions, recA803 should also suppress them by circumventing the interference. To perform the second test, an assay of joint-molecule formation catalyzed by RecA protein can be used. In this assay, part of a circular single-stranded (ss) D N A molecule displaces its counterpart from one end of an homologous linear duplex (ds) D N A molecule forming a plectonemic joint between the two molecules (Cox and Lehman 1987). Failure of mutant Ssb proteins to stimulate this reaction or inhibition of this reaction by mutant Ssb proteins would be consistent with a direct role of Ssb protein in recombinational repair of UV damage in vivo. Joint-molecule formation is a two-step process. Ssb protein is already known to affect the first step, which is formation of a RecA-ssDNA intermediate, called a presynaptic complex. Ssb (wt) protein added after RecA protein stimulates formation of presynaptic complexes (Cox and Lehman 1987). Added before RecA protein,

130 however, Ssb(wt) protein inhibits formation of this intermediate (Cox and Lehman 1987). This difference can be explained if one makes the following assumptions (1) the RecA-ssDNA presynaptic complex must include all portions of the DNA; (2) Ssb(wt) protein, when added before RecA protein, prevents RecA protein from forming the complex; and (3) Ssb(wt) protein, when added after RecA protein, renders regions of ssDNA available for RecA-ssDNA complex formation that are otherwise unavailable. Egner et al. (1987) has shown that SsbI protein fails to stimulate joint-molecule formation when added after RecA(wt) protein. This finding is consistent with a role of Ssb protein in recombinational repair and it provides a possible explanation of the defects in recombinational repair suffered by an ssb-1 mutant. Because Egner et al. (1987) have found that Ssb-113 protein acts like Ssb(wt) protein in stimulating jointmolecule formation, the possibility remains open that Ssb-113 may affect recombinational repair by interfering with RecF protein in vivo. This report describes our application of the suppressor and enzymatic tests to learn more about the role of Ssb protein in vivo. First we look at the UV sensitivity and ability to derepress the lexA regulon of recAS03 derivatives of ssb-1 and ssb-ll3 strains to assess the degree and nature of suppression. We then examine the ability of Ssb mutant proteins to inhibit joint-molecule formation when added before RecA protein. Finally, we compare the abilities of RecA(wt) and RecA803 proteins to overcome effects of Ssb mutant proteins. Materials and methods

Bacterial strains and P1 transductions. Strains used for determination of UV sensitivity were KH-12 derivatives (Bachmann and Low 1980). SR996 is recA +ssb+; SR997 is recA+ssb-ll3; SR999 is recA+ssb-1 (Wang and Smith 1984). These strains also carried leuB19 metE70 thyA36 bioA2 deo(c2) lacZ53 rha-5 rpsl-151 IN(rnhD-rrnE)F as markers. JC10236 is recA + srlC3OO::TnlO (Willis et al. 1981); MV1202 is recB21 recC21 sbcB15 sbcC201 recF143 recA803 (Volkert and Hartke 1984; Madiraju et al. 1988); JC8111 is recB21 recC21 sbcB15 sbcC201 recF143 (Horii and Clark 1973), and these strains are AB1157 derivatives. We transduced recA803 into SR996, SR997 and SR999 as described below. Our strategy was first to introduce the transposon TnlO, which confers tetracycline resistance (Tetr), into srIC close to recA803 and then contransduce recA803 and the TnlO into the recipient strains. Because the cotransduction frequency between recA and srlC has been found to be more than 95% (Csonka and Clark 1979), we had a high probability of losing recA803 in our construction of the srIC3OO::TnlO recA803 strains. We therefore used a recipient strain, MV1202 (see above), in which recA803 will provide UV and mitomycin C resistance by suppression of recF143 (Volkert and Hartke 1984). Further, MVI202 is sensitive to tetracycline. A lysate of Plvir was initially prepared on JC10236. MV1202 exposed to this lysate produced Tet r transductants, which were

screened for UV resistance (i.e., retention of recA803). Those transductants that retained the recA803 mutation were UV resistant. The ratio between Tet r UV r transductants and Tet r UV s transductants under these conditions is 4:96. One of the TetrUV r transductants was called MVM1. The recA803 genotype was confirmed by PI transductional back-crossing into JC8111 and testing for UV resistance among Tet r transductants. MVMI was used as P1 donor to transduce recAS03 into ABl157. One of the transductants is called MVM10. Either MVM1 or MVM10 was used as a PI donor to transduce SR996, SR997 and SR999 with recAS03. The recA803 derivatives are called MVM25, MVM26 and MVM27 respectively. UV response. Methods for determining UV resistance on L medium were as previously described (Rothman and Clark 1977). [Lgalactosidase assays. We transformed SR996, SR997 and SR999 and their transductants with pSE200 (Elledge and Walker 1983; originally obtained from M. Volkert) to test the effect of the recA803 mutation on UV-induced expression of lacZ fused to mucB which is itself under lexA control. Permeabilization of cells and assays of/?galactosidase levels were performed essentially as described by Miller (1972). Sources of proteins. RecA803 and RecA(wt) proteins were purified as described (Madiraju et al. 1988). Ssb(wt) protein was a generous gift from Stephen Kowalczykowski of Northwestern University, Chicago. Ssb-I and Ssb-113 proteins were kindly provided for use by John W. Chase of Albert Einstein College of Medicine, New York. Joint-molecule formation. Joint-molecule formation reactions were performed essentially as described by Madiraju et al. (1988). We prepared a reaction mixture containing 20 mM TRIS-HC1, pH 7.5, 10 raM, MgClz, 0.1 mM DTT, 0.1 mM EDTA, 3 gM ~X174 ssDNA, 1.0 gM Ssb protein. All samples were incubated at 34° C for 5 rain. At the end of the incubation period, RecA protein (1 gM final concentration) was added and incubations were continued either for 10 min for Ssb-ll3 protein or for 2 min for Ssb-1 protein. After the indicated incubation period, reactions were started by adding linear dsDNA (6.5 gM final concentration). Samples were removed at various times and processed to detect joint-molecules as described (Madiraju et al. 1988). An ATP regenerating system, containing phosphoenolpyruvate, pyruvate kinase, lactate dehydrogenase and N A D H was included in all assays. A TPase activities. The hydrolysis of ATP was monitored using a convenient spectrophotometric assay that couples the regeneration of ATP to the oxidation of N A D H in a mixture consisting of pyruvate kinase, phosphoenol pyruvate, lactate dehydrogenase and NADH. This method was originally described by Kreuzer and Jongeneel (1983) and has been adapted for use with RecA(wt) pro-

131

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+

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A

I

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10

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Fig. 1. Suppression of UV sensitivity by recA803 in ssb mutant backgrounds. Exponentially grown cultures of ssb mutants and their transductants were centrifuged from L broth, washed once

with minimal medium and exposed to the required UV doses. Survival rates of exposed cultures were determined after appropriate dilution

=

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m 1250 tein by Kowalczykowski and Krupp (1987). In this reaction oxidation of N A D H to N A D + is coupled to the regeneration of ATP from ADP. Rates of decrease in absorbance are thus directly proportional to rates of A T P hydrolysis.

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ssb- 113recA + ssb- 113recA803 F

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Results I

U V sensitivity

Survival rates of cell populations upon exposure to UV radiation are shown in Fig. 1, confirming previously published results that the reeA + ssb mutant strains are more UV sensitive than the wild-type strains. Our double mutants, recA803 ssb-1 and recA803 ssb-ll3, are more UV resistant than the recA + ssb-1 and recA + s s b - l l 3 strains (Fig. 1). The restoration of UV resistance by the recA803 mutation does not reach wild-type levels (data not shown), indicating only partial suppression. The recA803 mutation alone, by contrast, confers no more UV resistance than reeA + (data not shown). As measured by increased cell survival, the extent of the UV resistance recovered in the ssb-ll3 strain due to the recA803 mutation is more than 300-fold while that recovered in the ssb-i strain is only 10-fold. The survival of the recA803 mutants could be due either to increased recombinational repair ability of cells, or to increased UV derepression of LexA-controlled genes, which may facilitate other kinds of repair (e.g. excision). To test whether the recA803 mutation is suppressing any regulatory defect caused by the ssb mutant alleles, we transformed ssb mutants, both recA + and reeA803, with pSE200 DNA. This plasmid contains the

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40 2 4 Time (h) after UV (noUV t~ [] ; w [ t h U V ~ ] Fig. 2A-F. Expression of fl-galactosidase activity of transformants containing pSE200 upon UV irradiation. Overnight cultures of the strains of ssb mutants and their double mutants grown in L broth were diluted 100-fold in minimal medium supplemented with glucose, Casamino acids, leucine, biotin and thymine and grown to exponential phase (30 Klett units). Cells were then centrifuged, resuspended in the same medium and UV irradiated. At various times samples were removed, permeabilized with chloroform and SDS and fl-galactosidase activity assayed essentially as described earlier (Madiraju et al. 1988)

mucB promoter (mucBp) to which IacZ has been fused. Transcription from mucBp has been found to be LexAcontrolled (Elledge and Walker 1983) and can be followed very conveniently (e.g., Madiraju et al. 1988). Our results indicate that the presence of recA803 in the ssb wild-type background has no effect on UV-induced lacZ expression (Fig. 2A, B). Further, recA803 did not increase UV-induced transcription from mucBp to any great extent in ssb-1 and s s b l l 3 backgrounds (Fig. 2 C F). These results make it seem unlikely that recA803 suppresses the regulatory defects of ssb-i and ssb-ll3.

132 Ssb protein added Wild type

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Hg. 3A-F. Joint-molecule formation reaction catalyzed by RecAS03 and RecA(wt) proteins. Reaction samples containing complexes of Ssb, ssDNA and ATP in buffer were incubated with RecA(wt) protein or RecA803 protein as indicated. At the end

of the incubation period, linear dsDNA was added to start the reaction. Reactionsamplesat various timeintervalswere processed for joint-molecule formation as described in Materials and methods. P, protein; Ssb-lp, Ssb-1 protein, etc

Hence, we conclude that the partial suppression of the

times greater than 6 min (Fig. 3D, E). Ssb-1 protein was unable to inhibit RecA(wt) protein (Fig. 3A, C) under the less rigorous conditions, hence, data for more rigorous conditions are not shown. By contrast, Ssb-ll3 protein inhibited RecA(wt) protein slightly under the more rigorous conditions at earlier reaction times and failed to stimulate the reaction at late reaction times (Fig. 3 D, F). Under all these conditions RecA803 protein showed higher reactivity than RecA(wt) protein. It is interesting to note that Ssb-113 protein inhibited the joint-moleculeforming ability of RecA803 protein more than that of RecA(wt) protein at early reaction times (up to 2 rain, compare Fig. 3 E and F). From 4 to 10 rain however, RecA803 protein continues to catalyze joint-molecule formation while the reaction with RecA(wt) protein stops (Fig. 3F). In 10 rain, as many joint molecules are made by RecAS03 protein in the presence of Ssb-113 as in the presence of Ssb(wt) protein (Fig. 3 E, F).

ssb mutations by recA803 is probably due to suppression of a recombinational defect and hence an increase in recombinational repair.

Joint-molecule formation To investigate the role of Ssb mutant proteins in recombination, we studied their effect on the formation of joint-molecules in vitro. We first allowed complexes of ssDNA and Ssb protein to form in the presence of ATP by incubating the mixture at 34° C for 5 rain. Then RecA(wt) or RecAS03 protein was added and allowed to interact for either 2 or 10 rain, before linear dsDNA was added to initiate the reaction. Control reactions were carried out under identical conditions except that Ssb protein was omitted from the reaction mixture. The results show that when RecA(wt) protein was given only 2 rain to interact with ssDNA-Ssb(wt) protein complexes, substantial inhibition of joint-molecule formation occurred (Fig. 3 A, B) confirming what was already known (e.g., Madiraju et al. 1988). More rigorous conditions (10 min of interaction) not only alleviated the inhibition, but stimulated the reaction slightly at reaction

A TP hydrolysis and the formation ofpresynaptic complexes Inhibition by Ssb protein of joint-molecule formation could reflect inhibition of formation of presynaptic corn-

133

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plexes. To try to measure the amount of presynaptic complexes formed, without using joint-molecule formation to assay them, we turned to the ssDNA-dependent ATPase reaction. Our results indicated that in the absence of any added Ssb protein, both RecA(wt) and RecA803 proteins efficiently catalyzed ATP hydrolysis, though RecA803 protein showed both a higher steady state rate than RecA(wt) protein (Fig. 4A) and attained this rate faster. In the presence of Ssb(wt) or Ssb mutant proteins, the ATP hydrolysis activity catalyzed by RecA(wt) protein was inhibited significantly or completely for up to 1400 s (Fig. 4B-D). Unlike RecA(wt) protein, the mutant RecA803 protein catalyzed ATP hydrolysis significantly in the presence of Ssb protein though the rates were somewhat lower than in the control reactions where no Ssb protein was added. Under these conditions ATP hydrolysis did not reach a steady state rate. In order to quantitate the extent of the observed inhibition, we compared the ATPase activities retained at early (200 s) and late (1000 s) reaction times. These values (Table 1) show that both RecA proteins were inhibited by wild-type Ssb protein but that wild-type RecA protein was inhibited 10 and 7 times as much as RecA803 protein at early and late times respectively. Both mutant Ssb proteins inhibited the ATPase activities of the RecA proteins to about the same extent as the wild-type Ssb protein (Table 1).

1000

1500

Table 1. ATPase activities retained by RecA proteins when added after Ssb proteins to ssDNA

Ssb protein added

Early (200 s)

Late (1000 s)

RecA(wt) RecAS03 RecA(wt) RecA803 protein p r o t e i n p r o t e i n protein None 100 Ssb(wt) protein 3 Ssb-1 protein 3 Ssb-ll3 protein 1

100 30 31 28

100 11 6 9

100 76 78 68

One feature of these results indicates that ssDNAdependent ATPase activity may not be a valid measure of presynaptic complexes. The degree of inhibition of the joint-molecule reaction by Ssb wild-type protein varies with the time allowed for RecA wild-type protein to interact prior to adding dsDNA. If 2 min is allowed (Fig. 3 B), joint-molecule formation is almost completely inhibited; if 10 min is allowed (Fig. 3 E), there is no inhibition. By contrast, Fig. 4 shows that at times up to 1400 s (>23 min), RecA wild-type protein is unable to overcome inhibition at the ATPase reaction by Ssb wildtype protein (Fig. 4B). One possible explanation of this difference is that an inhibitor is present in the ATPase reaction but not in the joint-molecule reaction. Since

134 N A D H , N A D and lactic dehydrogenase are present in the ATPase but not in the joint-molecule reaction, we tested those materials for their ability to inhibit jointmolecule formation. No significant inhibition was observed under the conditions employed in Fig. 4B (data not shown). Another possibility is that something present in the joint-molecule reaction helps to overcome inhibition by Ssb wild-type protein. In this case the only differentiating material is dsDNA. Consequently, we checked the effect on the ATPase reaction of adding d s D N A under the conditions employed in Fig. 4B. We found that ssDNA-dependent ATPase activity is stimulated by the presence of d s D N A (data not shown). This suggests that homologous pairing or synapsis may overcome the inhibitory effects o f Ssb protein. Tests of this possibility will be reported elsewhere.

Table 2. Comparisons between ATPase activity and joint-moleculeformating ability of RecA(wt) protein and RecA803 protein A Sample

No Ssb protein Ssb(wt) protein Ssb-1 protein

RecA(wt) protein Time after addition

RecA803protein Time after addition

%JM" 5 rain

ATPase b 7 rain

%JM" 5 rain

ATPase b 7 min

15 1 16

0.00060 0.0000/ 0.00001

59 39 64

0.00100 0.00040 0.00032

a Since the reaction mixture was preincubated with RecA protein for 2 min before the addition of linear dsDNA, the 5 rain jointmolecule formation time represents a total of 7 min contact time of RecA protein with ssDNA. Hence, this value is compared with 7 rain (420 s) ATP hydrolysis rate b ATP hydrolysis is represented as rate/sec

Discussion We have examined the possibility that ssb mutations exert their mutant effects on UV resistance and repair by interfering with the normal functions of RecF protein. The suppressor test showed that recA803 was able partially to circumvent the effects o f both ssb-1 and ssb-ll3 on UV sensitivity. Because RecA803 did not alter the low level of derepression o f the lexA regulon caused by the ssb mutations, we infer that the ssb mutant effects suppressed by recA803 involve recombinational repair. The ability of recA803 to act on repair without influencing regulation o f the lexA regulon, originally found by Madiraju et al. (1988), confirms that RecA protein plays a direct role in repair as well as an indirect regulatory role. Such a direct role could also be inferred from the work of M o u n t et al. (1975) who showed that derepressing the lexA regulon of a recA1 strain increased excision repair but did not restore UV resistance completely. From our enzymatic work, there is no reason to expect that the effects of ssb-1 on recombinational repair are exerted by inhibition of RecF protein in vivo. In ssb-1 His(55) is changed to Tyr (Chase and Williams 1986). This decreases the stability o f the tetrameric form of Ssb protein, which seems to be the active form in vivo. Williams et al. (1984) have shown that increasing the in vivo concentration o f Ssb-1 protein suppresses the effects of the mutation. Presumably tetramers of Ssb-1 protein are fully active and when formed in sufficient quantity can carry out all the functions of wild-type Ssb protein. We did not observe inhibition by Ssb-1 protein of either joint-molecule formation or ATPase under conditions that did not allow inhibition by Ssb(wt) protein. It is therefore plausible to correlate the UV sensitivity defect, suppressible by recA803 with the failure of Ssb-I protein to stimulate joint-molecule formation by RecA(wt) protein (Egner et al. 1987). Accordingly, suppression by recA803 can be correlated with the greater in vitro activity of RecA803 in forming joint-molecules in the absence o f Ssb protein (Madiraju et al. 1988). Consequently, there is no necessity to invoke an interference of Ssb-1 protein with RecF protein to explain the mutant effects on recombinational repair.

Sample

No Ssb protein Ssb(wt) protein Ssb-I protein

RecA(wt) protein Time after addition

RecA803protein Time after addition

%JM a 5 rain

ATPase b 15 rain

%JM a 5 rain

ATPase b 15 rain

41 40 30

0.00073 0.00001 0.00002

78 84 52

0.00100 0.00074 0.00062

" Since the reaction mixture was preincubated with RecA protein for 10 rain before the addition of linear dsDNA, the 5 rain jointmolecule formation time represents total of 15 rain contact time of RecA protein with ssDNA. Hence, this value is compared with 15 rain (900 s) ATP hydrolysis rate b ATP hydrolysis is represented as rate/sec

In ssb-ll3 Pro(176) is changed to Ser (Chase and Williams 1986). This increases the binding affinity of the mutant protein for ssDNA. Chase et al. (1984) have shown that increasing the in vivo concentration of Ssb113 protein did not restore the wild-type phenotype. Presumably, therefore, Ssb-113 protein is intrinsically defective in normal function or, as suggested in this paper, it interferes with the normal funcition of RecF protein. We did find one in vitro correlate with the UV sensitivity of the ssb-ll3 mutant and its partial suppression by recA803. Comparison of the data in Fig. 3E and F shows that Ssb-ll3 protein slightly inhibits the rate of joint-molecule formation by RecA(wt) protein and the final yield at 10 min. Although Ssb-ll3 protein also inhibits the rate of joint-molecule formation by RecA803 protein, the final yield of joint-molecules is the same as with Ssb(wt) protein. This implies that the joint-molecule-forming ability of RecA803 protein is less inhibited than that of RecA(wt) protein. Whether or not this phenomenon provides a sufficient rationale for the recA803suppressible component o f UV sensitivity requires evaluation by further in vitro experiments. Such experiments require a larger supply o f Ssb-113 protein than was available at the time this work was performed. We would,

135 however, like to direct attention to the possibility that in vivo Ssb-113 protein interferes with R e c F protein function. I f r e c F m u t a t i o n s could be f o u n d that suppress s s b - I 1 3 , it w o u l d s u p p o r t this hypothesis. O u r in vitro w o r k also p r o d u c e d two observations concerning the relation between the s s D N A - d e p e n d e n t A T P a s e activity o f R e c A protein and its joint-moleculef o r m i n g activity, Firstly, we s h o w e d that the two are differentially inhibitable by Ssb proteins (Table 2 A , B). The role o f d s D N A in this differential inhibition will be r e p o r t e d elsewhere. Secondly, we can see a possible w a y to study h o w the f o r m o f Ssb protein affects its a n t a g o n i s m to R e c A protein. At 1 g M , the concentration o f Ssb protein t h a t we employed, Ssb-1 protein is p r e d o m i n a n t l y in the m o n o m e r i c f o r m while Ssb(wt) protein is mainly in the tetramic f o r m (Williams et al. 1984). Because Ssb-1 protein at this c o n c e n t r a t i o n did n o t inhibit joint-molecule f o r m a t i o n by RecA(wt) protein, while Ssb(wt) protein did, we can infer that tetramers, n o t m o n o m e r s , inhibit this reaction. Because b o t h Ssb proteins inhibited the A T P a s e reaction, there are two possibilities: (1) b o t h m o n o m e r s a n d tetramers can inhibit; or (2) a low c o n c e n t r a t i o n o f tetramers is sufficient to inhibit the A T P a s e . F u r t h e r study o f these possibilities also requires a larger a m o u n t o f Ssb-1 protein t h a n was available when this study was undertaken. Acknowledgements. We thank Dr. Stephen C. Kowalczykowski for

supplying Ssb protein, Dr. John Chase for the mutant Ssb-1 and Ssb-113 proteins, Dr. T.V. Wang for strains, Ann Templin for teaching M.V.V.S.M. transductional crosses, Dr. Raymond Devoter for his interest in this work. Drs. S.C. Kowalczykowski and S. Sandler for comments on the manuscript and Nelle NeighborAlonzo for carefully typing the manuscript. This work was supported by NIH research grant no. AI05371 from the Institute of Allergy and Infectious Diseases.

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