Escherichia coli recA protein protects single-stranded DNA or gapped duplex DNA from degradation by RecBC DNase.

RecA- mutants of Escherichia coli extensively degrade their DNA following UV irradiation. Most of this degradation is due to the recBC DNase, which suggests that the recA gene is involved in the control of recBC DNase in vivo. We have shown that purified recA protein inhibits the endonuclease and exonuclease activities of recBC DNase on single-stranded DNA. The extent of inhibition is dependent on the relative concentration of recA protein, recBC DNase, and the DNA substrate; inhibition is greatest when the concentrations of DNA and recBC DNase are low and the concentrations of recA protein is high. At fixed concentrations of recA protein and recBC DNase, inhibition is eliminated at high concentrations of DNA. In the presence of adenosine 5'-O-(3-thiotriphosphate), an ATP analog which stabilizes the binding of recA protein to both single- and double-stranded DNA, recA protein is a more potent inhibitor of the nuclease activities on single-stranded DNA and is a weak inhibitor of the exonuclease activity on double-stranded DNA. Inhibition of the latter is enhanced by oligodeoxynucleotides, which stimulate the binding of recA protein to double-stranded DNA. In the presence of adenosine 5'-O-(3-thiotriphosphate), recA protein also inhibits the action of exonuclease I on single-stranded DNA and of lambda exonuclease on double-stranded DNA. These observations are most consistent with the idea that recA protein protects DNA from recBC DNase by binding to DNA. RecA protein also blocks the endonucleolytic cleavage of gapped circular DNA by recBC DNase. Since both recA protein and recBC DNase have the ability under certain conditions to unwind duplex DNA and to displace strands, we looked for evidence that their combined action would enlarge gaps but found no extensive enlargement. D-loops, a putative intermediate in genetic recombination, are effectively protected against the action of recBC DNase by the E. coli single strand binding protein and by recA protein in the presence of adenosine 5'-O-(3-thiotriphosphate).

RecAmutants of Escherichia coli extensively degrade their DNA following W irradiation. Most of this degradation is due to the recBC DNase, which suggests that the recA gene is involved in the control of recBC DNase in vivo. We have shown that purified recA protein inhibits the endonuclease and exonuclease activities of recBC DNase on single-stranded DNA. The extent of inhibition is dependent on the relative concentrations of recA protein, recBC DNase, and the DNA substrate; inhibition is greatest when the concentrations of DNA and recBC DNase are low and the concentration of recA protein is high. At fixed concentrations of recA protein and recBC DNase, inhibition is eliminated at high concentrations of DNA. In the presence of adenosine 5'-0-(3-thiotriphosphate), an ATP analog which stabilizes the binding of recA protein to both single-and double-stranded DNA, recA protein is a more potent inhibitor of the nuclease activities on single-stranded DNA and is a weak inhibitor of the exonuclease activity on double-stranded DNA. Inhibition of the latter is enhanced by oligodeoxynucleotides, which stimulate the binding of recA protein to doublestranded DNA. In the presence of adenosine 5'-0-(3thiotriphosphate), recA protein also inhibits the action of exonuclease I on single-stranded DNA and of X exonuclease on double-stranded DNA. These observations are most consistent with the idea that recA protein protects DNA from recBC DNase by binding to DNA. RecA protein also blocks the endonucleolytic cleavage of gapped circular DNA by recBC DNase. Since both recA protein and recBC DNase have the ability under certain conditions to unwind duplex DNA and to displace strands, we looked for evidence that their combined action would enlarge gaps but found no extensive enlargement. D-loops, a putative intermediate in genetic recombination, are effectively protected against the action of recBC DNase by the E. coli single strand binding protein and by recA protein in the presence of adenosine 5'-0-(3-thiotriphosphate).
The recA and recBC genes in Escherichia coli K12 play an important role in cell viability, in genetic recombination, and in the repair of damaged DNA.
Mutations in the recA gene cause a reduction in genetic recombination by as much as IO-", high sensitivity to UV and x-irradiation, loss of UV mutability, and extensive "reckless" degradation of DNA following UV or x-irradiation (Clark, 1973). Only half of the cells in a recAculture are viable; the dead cells contain no DNA (Capaldo and Barbour, 1975). The recA gene product is a protein of 37,800 daltons (Sancar et al., 1980, Horii et al., 1980 which has been shown to have at least two distinct functions. First, in the presence of ATP and single-stranded DNA, it acts as a protease, inactivating the ZexA repressor (Little et al., 1980) as well as the phage X repressor (Roberts et al., 1978a). The lexA repressor is involved in the expression of several genes affecting cell division, mutagenesis, and DNA repair ("SOS' repair) (Witkin, 19761, including the recA gene (Mount, 1977). Thus, recA regulates its own expression; in a fully induced cell, recA protein constitutes up to 3% of the total protein (Gudas and Mount, 1977). SOS induction is triggered by agents which damage DNA or block replication, an event thought to lead to the appearance of a "signal molecule," such as single-stranded DNA, which in turn activates the protease activity of recA protein (Little et al., 1980). Second, recA protein is a DNAdependent ATPase (Ogawa et al., 1978;Roberts et al., 1978b) which catalyzes the joining of DNA molecules at homologous sites and assimilates single strands into double-stranded molecules in a heteroduplex joint (Weinstock et al., 1979;Shibata et al., 1979a;DasGupta et al., 1980). This property is interesting with regard to the role of recA in genetic recombination.
RecBC DNase consists of two subunits of 130,000 and 140,000 daltons (Goldmark and Linn, 1972). RecBC DNase is absent from recBand r e dmutants and there is evidence that the recB gene encodes one of the subunits of the DNase (Tomizawa and Ogawa, 1972;Lieberman and Oishi, 1974). RecBC DNase cleaves single-stranded DNA endonucleolytic d y and degrades both single-and double-stranded DNA exonucleolytically. The endonuclease is stimulated 7-fold by ATP, whereas the exonuclease activities require ATP, hydrolyzing more than 20 ATP molecules for each phosphodiester bond cleaved (Goldmark and Linn, 1972). RecBC DNase also has an ATP-dependent unwinding activity which, in the presence of Ca", transiently denatures long stretches of doublestranded DNA, occasionally nicking one strand or the other as the enzyme moves along the DNA (Rosamond et al., 1979, Taylor and. Genetic recombination in recBor re&mutants is affected to a varying degree depending on the type of recombination measured, ranging from 0.3 to 60% of the rect level (Hall and Howard-Flanders, 1972;Clark, 1973). Although one role of recBC DNase in recombination may be to promote cell viability following a recombination event (reviewed in Radding, 1978), Kolodner (1980) has shown that recBC DNase stimulates recombination when added to a recombination-deficient extract prepared from recB-re&-recFcells, which suggests that recBC DNase may be directly involved in some kinds of recombination. Only one-fourth of 7573 RecA Protein Protects DNA from Degradation the cells in a recB-or r e c C culture are viable (Capaldo and Barbour, 1975), and the viable cells are about 3-fold more sensitive than wild type cells to UV irradiation (Willetts and Clark, 1969). A recB270 recC271 double mutant is rec+ at 30 "C but is phenotypically recBC-at 43" C. RecBC DNase purified from this strain is abnormally thermolabile only in the ATP-dependent exonuclease activity on double-stranded DNA, which suggests that this activity may be of special importance in viuo (Kushner, 1974). RecBC DNase is required for the induction of recA protein following treatment with nalidixic acid but not with UV light (Gudas and Pardee, 1976;McPartland et al., 1980). A relationship between DNA degradation by recBC DNase and SOS induction is indicated by the experiments of Oishi and Smith (1978), in which the inactivation of phage @O repressor in permeabilized cells was correlated with the degradation by recBC DNase of newly replicated DNA at stalled replication forks. DNA degradation is also a consequence of excision repair, in which damaged bases are removed along with undamaged nucleotides in the same strand and replaced by new nucleotides using the undamaged strand as template (Hana-Walt, 1975).
Although DNA degradation is a normal part of DNA metabolism, extensive reckless degradation occurs in recA-mutants following UV irradiation (Clark et al., 1966). The extensive degradation seen in a recA-mutant is not observed in a recA-recB-double mutant, which suggests that recA protein either directly or indirectly controls the action of recBC DNase (Willetts and Clark, 1969). Satta et al. (1979) suggested that recA protein protects DNA from recBC DNase in viuo by binding to single-stranded DNA. Their experiments utilized a set of lexA-mutants which produce recA protein at different rates following treatment with nalidixic acid. Chromosomal DNA was degraded in response to treatment with nalidixic acid, and the rate of degradation in each mutant was inversely proportional to the rate of production of recA protein. Degradation of newly replicated DNA at replication forks was not affected by the level of recA protein, however.
RecA protein and recBC DNase are not the only two enzymes involved in DNA degradation in E. coli. Excessive degradation following x-irradiation has been reported in ras-, polA-, and uurD-strains, as well as in recA-mutants. In these strains, 75 to 90% of the degradation is accounted for by recBC DNase, with the remainder attributed to other nucleases (Youngs and Berstein, 1973).
The availability of purified recA protein (Shibata et al., 1981a) and recBC DNase (Goldmark and Linn, 1972) has made it possible to study the interaction between recA protein and recBC DNase on a variety of DNA substrates in uitro. In this paper we report the results of studies on the inhibition of recBC DNase by purified recA protein.
Agarose Gel Electrophoresis-For electrophoresis, we used a vertical slab gel apparatus (17 X 16 X 0.3 cm; Blair Craft, Cold Spring Harbor, NY). The running buffer was 40 mM Tris-acetate, pH 7.9, 5 mM Na-acetate, 1 mM EDTA. After electrophoresis, gels were stained in ethidium bromide (0.5 pg/ml) and the DNA was visualized under ultraviolet light.
Endonuclease Assay-The assay measures the conversion of single-stranded circular DNA to linear molecules which can be degraded by exonuclease I to acid-soluble nucleotides. After endonucleolytic cleavage by recBC DNase, 2 0 4 samples were mixed with 200 p1 of 70 mM glycine-NaOH, pH 9.5, 2 mM EDTA, 200 p1 of chloroform. After shaking for 1 min and centrifuging in a Beckman microfuge, 150 pl of the aqueous (top) phase were recovered and heated in a glass tube at 60 "C for 5 min. The sample was chilled on ice and mixed with 40 p1 of 50 mM MgCL and 20 units of exonuclease I in 10 p1 of diluent (50 mM Tris-HC1, pH 7.5, 250 m~ ammonium sulfate, 1 mg of BSA/ml). Incubation was at 37 "C for 15 min. Acid-soluble DNA was measured as described below.
Determination of Acid-soluble Nucleotides-Unless otherwise noted, the following procedure was used to measure acid-soluble nucleotides. The sample was diluted to a final volume of 200 pl by the addition of 25 mM EDTA. Acid-soluble DNA was determined by mixing a 150-p1 aliquot of each sample with 350 p1 of calf thymus DNA (1 mg/ml) and adding 500 pl of 10% trichloroacetic acid. After incubation at 0 "C for 10 min, the precipitate was removed by centrifugation at 10,000 rpm for 5 min a a Sorvall SE-12 rotor at 4 "C.
The supernatant (800 pl) was counted in 7 ml of Triton fluor. Total DNA in each sample was determined by mixing, in this order, a 30-p1 aliquot of sample, 370 p1 of water, 7 ml of Triton fluor, and 400 pl of 10% trichloroacetic acid in a scintillation vial and counting.
D-loop Assay-This assay measures the retention of D-loops by a nitrocellulose filter under conditions in which single-stranded DNA, but not double-stranded DNA, binds to the filter (Beattie et al., 1977).
The sample was diluted with 25 m~ EDTA, pH 8.0, to a final volume of 300 pl. A 2 0 0 4 aliquot was mixed with 1 ml of 1.5 M NaCV0.15 M Na-citrate (NaCl/Na-citrate), pH 7.0, incubated a t 41 "C for 4 min, mixed with 6 ml of NaCl/Na-citrate a t 0 "C, and passed through a nitrocellulose filter at a flow rate of 4 m1/10 s. The filter was washed with 8 ml of NaCl/Na-citrate. Total DNA in each sample was determined by spotting a 50.~1 aliquot on a filter. The filters were dried under a heat lamp and radioactivity on the filters was measured by scintillation counting.
DNA-DNA concentrations are given in terms of the molarity of nucleotides. Published procedures were used for the preparation of +X174am3 phage DNA and form I DNA: and of fd phage DNA (Cunningham et al., 1980). E. coli ["HIDNA was prepared by the method of Marmur (1961) from E. coli HF4704 grown to stationary phase in TPG medium (Cunningham et al., 1980) containing thymine (2 pg/ml) and [methyl-'HH] thymidine (5 pCi/ml). Mu DNA was prepared as described elsewhere (Williams and Radding, 1981). Oligodeoxynucleotides, a limit digest of salmon sperm DNA by pancreatic DNase, were prepared as described (Cunningham et al., 1979).
Gapped Circular DNA--"*P-labeled double-stranded fragments of +X174am3 DNA were prepared by digesting +X174am3 form I ["PIDNA to completion with restriction endonuclease Hue 111. The 2-ml reaction mixture contained 5.6 mM Tris-HC1, pH 7.5, 6.6 mM NaC1,6.6 mM 2-mercaptoethanol, 1 mM MgCIz, 50 p~ EDTA, 110 pg of BSA/ml, 202 PM 4X174am3 form I ["PIDNA, 525 units (New England Biolabs) of Hue 111. Incubation was at 37 "C for 3 h. The reaction mixture was extracted twice with phenol and the DNA was precipitated by the addition of 1/5 volume of 1.8 M Na-acetate, pH 7.0, (final concentration = 0.3 M), 3 volumes of 95% ethanol. The DNA was held at -20 "C for 12 h and the precipitate was recovered by centrifugation at 25,000 rpm for 20 min in a Beckman SW27 rotor. The pellet was dissolved in 250 pl of 10 m~ Tris-HC1, pH 7.5, 0.1 mM EDTA and dialyzed against 1 liter of this buffer for 12 h. The DNA fragments were denatured at 70 "C for 5 min in a volume of 780 ~1 that contained 225 p~ fragments, 25 mM Tris-HCI, pH 8.0, 0.3 M NaCI, 30 mM sodium citrate, pH 7.0, 49.5% formamide. +X174am3 Form I DNA is the closed double-stranded circular form of phages 4x174 and fd; circular DNA with one or more single-stranded breaks is termed form 11. phage ['HIDNA was added in a volume of 14 p1 to a final concentration of 37.5 PM. The reaction mixture was transferred from 70 "C to a 37 "C water bath and incubated for 12 min. It was then dialyzed against 10 mM Tris-HC1, pH 7.5, 10 mM NaCI. 1 mM EDTA for 5 h a t room temperature. The DNA was layered onto a 17-ml 5-20% sucrose gradient (in 10 mM Tris-HC1, pH 7.5, 10 mM NaCI, 1 r n~ EDTA) and centrifuged a t 27,000 rpm in a Beckman SW27.1 rotor a t 4 "C for 20 h. The fractions that contained "H ( Fig. 1) were pooled and the DNA was precipitated with ethanol. The precipitate was dissolved in 10 mM Tris-HCI, pH 7.5.0.1 mM EDTA and dialyzed against this buffer.
Characterization of gDNA-The hybridization mixture (see above) contained 175 nmol of ["'PIDNA and 29 nmol of ['HIDNA. Twelve per cent of the ["PIDNA, or 21 nmol, sedimented with the ["HIDNA ( Fig. 1). Thus, the average gDNA molecule is doublestranded along 72% of its length (21 nmol of ["?P]DNA + 29 nmol of ['HIDNA = 0.72). We further characterized gDNA by agarose gel electrophoresis. Lane 1 in Fig. 2 shows the restriction fragments of cpX174am3 form I DNA used in the preparation of gDNA. Lane 2 shows a mixture of +X174am3 forms I and 11; the faster migrating band is form I (Cunningham et al., 1979). The gDNA consists of two bands (lane 3). Cleavage of gDNA with SI nuclease (lane 4 ) resulted in a decreased amount of the slower migrating band and an increased amount of the faster migrating band, indicating that the slower band is circular DNA and the faster band is linear DNA. Digestion with increasing amounts of SI nuclease (lanes 5 and 6) resulted in the disappearance of both the circular and linear DNA and in the appearance of multiple bands. Only the ["HIDNA was degraded to acidsoluble nucleotides by SI nuclease (Fig. 2), which shows that only ["HIDNA was present as single strands; the [:'2P]DNA was in duplex form. The distribution of "H and . ' 21' in the lane containing only gDNA was determined by slicing the gel into 3-mm strips and counting these isotopes in each strip (Fig. 3). Eighty per cent of the "H was in the slower moving band of circular DNA, with the remainder in the band of linear DNA. Separation of the circular and linear forms was incomplete. The molar ratio of [:'ZP]DNA to [:'H]DNA across the circular band decreased from 0.85 to 0.48 in the direction of migration, which is consistent with the slower migration of circular duplex molecules that contain fewer or smaller gaps. D-loops-Fragments of single-stranded DNA were prepared by heating unlabeled cpX174am3 phage DNA at 100 "C for 22 min in 10 mM Tris-HC1, pH 7.5,O.l mM EDTA. Fragment length was between 300 and 600 nucleotides, as estimated by electrophoresis in a 1.1% agarose gel (not shown). For length standards, we used heat-denatured DNA (heated a t 100 OC for 2 min) from a Hue I11 digest of +X174arn3 form I (see preparation of gapped DNA). D-loops were prepared in a reaction volume of 500 pl that contained 500 p~ singlestranded fragments, 100 PM +X174arn3 form I ["HIDNA, 10 mM Tris-HCI, pH 7.5, 1 mM EDTA, 0.2 M NaCI. Incubation was at 75 OC for 30 min. The reaction mixture was layered onto a 17-ml 5-206 sucrose

RecA Protein
Protects DNA from Degradation reaction mixture (3.5 mi) contained 30 nm Tris-HC1, pH 7.5, 60 PM Mu [3H]DNA, X exonuclease (100 units/&). The reaction mixture was incubated at 25 "C for 5 min and digestion was initiated by the addition of 7 pl of 1 M MgCL Incubation was continued at 25 "C for 3 min and digestion was terminated by the addition of 140 pl of 250 mM EDTA. Acid-soluble DNA was determined as described above. The DNase made 2.8% of the Mu DNA acid-soluble, whereas 0.03% was acid-soluble before digestion. Since Mu DNA is 38,000 nucleotide pairs in length (38 kilobase pairs) (Allet et al., 1977), 2.8% digestion corresponds to 1,060 nucleotides removed from each end of the Mu DNA. The DNA was extracted with phenol, extracted with ether, precipitated with ethanol, and dissolved in 0.2 ml of 10 mM Tris-HCI, pH 7.5, 0.1 mM EDTA. The DNA was separated from acid-soluble nucleotides by gel filtration (Ultrogel AcA 34 equilibrated in 10 mM Tris-HCI, pH 7.5, 0.1 mM EDTA; column dimensions were 0.6 cm in inner diameter X 7 cm long).
Cleavage of Mu DNA by endonuclease Eco RI produces two terminal fragments (5 and 13 kilobase pairs) and one internal fragment (18 kilobase pairs) (Allet et al., 1977). When we digested tailed Mu DNA to completion (as shown by agarose gel electrophoresis) with Eco RI, 49% of the DNA was retained by a nitrocellulose filter in the D-loop assay (compared to 95% retention before cleavage and 470 retention of native Mu DNA before or after cleavage), and the 5 kilobase pair terminal fragment had a higher electrophoretic mobility than the corresponding fragment of native Mu DNA (not shown).
These results indicate that single-stranded DNA was present on both ends of tailed Mu DNA.
Enzymes-RecA protein was purified as described by Shibata et al. (1981a) and diluted in recA buffer (50 mM Tris-HC1, pH 7.5,5 mM dithiothreitol, 0.3 nm EDTA, 10% glycerol). Exonuclease I was purified from the overproducing strain SKI447 according to the procedure of Lehman and Nussbaum (1964). Under their conditions, 1 unit of exonuclease I degrades 10 nmol of single-stranded DNA to acidsoluble nucleotides in 30 min at 37 "C. The E. coli single strand DNA binding protein was a gift from Dr. M. Gefter (Molineux et al., 1974) and was heated at 100 "C for 10 min before use. RecBC DNase was purified 1500-fold from E. coli H560 by a modification of the procedure of Goldmark and Linn (1972). Assay conditions were 10 mM Tris-HC1, pH 7.9,lO mM 2-mercaptoethanol, 1 mg of BSA/ml, 10 mM MgS04, 0 or 220 p~ ATP, 15 p~ Mu ["'PIDNA in a volume of 100 pl. One unit of recBC DNase catalyzes the ATP-dependent conversion of 1 nmol of Mu DNA to acid-soluble nucleotides in 20 min at 37 "C. The most pure fraction had a specific activity of 14,700 units/mg of protein. Details of the purification and characterization of recBC DNase are described by Williams (1981). RecBC DNase was diluted in recBC diluent (10 mM Tris-HCI, pH 7.5, 1 mM dithiothreitol, 1 mg of BSA/ml). Proteinase K was purchased from EM Laboratories. Egg white lysozyme was purchased from Worthington. h exonuclease (phosphocellulose fraction a) was prepared and assayed as described by Radding (1971) and was a gift from Dr. C. DasGupta. Restriction endonucleases were purchased from Bethesda Research Laboratories, with the exception of endonuclease Hue I11 which was purchased from New England Biolabs. SI nuclease was purchased from Miles Laboratories.
Protein concentrations were determined by the method of Lowry et al. (1951) using BSA as the standard. The concentrations of recA protein (M, = 37,842) and SSB (Mr = 22,000) are expressed as moles of the monomeric polypeptide.

RecA Protein Protects Single-stranded
DNA from Exonucleolytic Attack by RecBC DNase-The ATP analog ATP+ competitively inhibits the DNA-dependent ATPase of recA protein and enhances the binding of recA protein to DNA (Shibata et al., 1979b); however, under the conditions of the present experiments, the analog does not affect the nuclease activities of recBC DNase. Therefore we were able to use ATPyS as an adjunct in our experiments on the interaction between recA protein and recBC DNase.
To study the effect of recA protein on the exonucleolytic protein efficiently inhibited the exonuclease activity, with maximum inhibition occurring at a ratio of 1 molecule of recA protein (M, = 37,800) t o 10 nucleotide residues of DNA, Inhibition was weaker in the absence of ATPyS, especially at the higher concentration of recBC DNase. The latter observation is consistent with either direct inhibition of the nuclease or with competition between recA protein and recBC DNase for substrate. The effect of ATPyS, which enhances the binding of recA protein to DNA (Shibata et al., 1979b), favors the second explanation.
T o s t u d y the mechanism of inhibition, we incubated fixed amounts of recA protein and recBC DNase with various amounts of single-stranded DNA and measured the production of acid-soluble nucleotides. The percentage of nuclease activity in the presence of recA protein was calculated by comparison to a control from which recA protein was omitted at each DNA concentration tested. In a l l reaction mixtures, DNA was in the excess so that the amount of DNA degraded was largely independent of the DNA concentratiom3 The inhibitory effect of recA protein was directly related to the ratio of recA protein to DNA (Fig. 5A). Inhibition was less effective at high concentrations of DNA, suggesting that inhibition is not caused by a direct interaction between recA protein and recBC DNase, since the concentrations of these two enzymes were held constant.
To estimate the ratio of recA protein to DNA at which inhibition was abolished, we plotted the data of Fig. 5A on double log coordinates and extrapolated the resulting straight lines to zero inhibition (i.e. 100% DNase activity). The inhibitory effect of recA protein is apparently eliminated at a ratio of 1 recA monomer/800 nucleotide residues (Fig. 5B).
These experiments (Figs. 4 and 5) are most easily interpreted to mean that recA protein binds to single-stranded DNA and protects it from recBC DNase. To demonstrate that tured E. Coli [,'%]DNA and recBC DNase, the latter at con-3 The amount of DNA made acid-soluble in the absence of recA centrations of 2 u n i t s / d o r 8 u n i t s / d , in the presence or protein increased from 34 to 48 pmol as the DNA concentration was absence of ATPyS (Fig. 4). I n the presence of ATPyS, recA increased from IO to 200 p~, respectively. was varied while the concentrations of recA protein and recBC DNase were held constant. Acid-soluble DNA was measured. On the ordinate is the percentage of acid-soluble DNA relative to a sample without recA protein a t each concentration of DNA. On the abscissa is the ratio of recA protein to nucleotide residues of DNA. The reaction mixtures (20 p l ) contained 10 mM Tris-HCI, pH 7.5, 1.5 mM dithiothreitol, 1.3 mg of BSA/ml, 10 mM MgC12, 1 mM ATP, 0 or 0.5 mM ATPyS, 10, 20, 50, 100, or 200 p~ heat-denatured E. coli ["HIDNA (see Fig. 4). 0 or 0.4 p~ recA protein, 0.04 unit of recBC DNase. Incubation was at 37 "C for 30 min. Acid-soluble DNA was measured as described under "Experimental Procedures." Acid-soluble backgrounds at each DNA concentration were determined from controls lacking both recA protein and recBC DNase. 0, without ATPyS; A, with ATPyS. B, the data from A plotted on double log coordinates.
recA protein binds to DNA under these conditions, we used an assay in which DNA is retained by a nitrocellulose filter only if it is complexed with recA protein (Shibata ef al., 1979b). RecA protein and heat-denatured E. coli ["]DNA were incubated in the presence of ATPyS under the same conditions used in the inhibition experiment of Fig. 5, except that recBC DNase was omitted. All of the DNA was retained by the filter at a ratio of recA monomer to nucleotide residues of DNA as low as 1:50, with retention decreasing to zero at a ratio of 1:500 (Fig. 6), which corresponds roughly to the least amount of recA protein required to protect t.he DNA from nuclease (see above).
Additional evidence in favor of protection of DNA via the binding of recA protein is provided by the effect of recA protein on exonuclease I. The degradation of single-stranded DNA by exonuclease I was strongly inhibited by recA protein in the presence of ATPyS, whereas inhibition was much weaker in the absence of ATPyS (Fig. 7). Thus, the inhibitory effects of both recA protein and ATPyS are not specific for recBC DNase, a result which is most easily explained if recA protein inhibits nucleases by binding to single-stranded DNA.
RecA Protein Protects Single-stranded DNA from Endonucleolytic Cleavage by RecBC DNase-We measured the endonucleolytic cleavage of single-stranded circular fd phage DNA by its conversion to a linear form that can be degraded by exonuclease I (see Goldmark and Linn, 1972). However, since recA protein inhibits exonuclease I (Fig. 7), it was necessary to inactivate both recA protein and recBC DNase by extraction with chloroform and heating at 60 "C before adding an excess of exonuclease I (WiLliams, 1981). As determined by gel electrophoresis (Williams, 1981), the circular DNA substrate used in these experiments contained some linear molecules, resulting in a 15% background of DNA that was sensitive to exonuclease I.
We incubated increasing amounts of recA protein with fd [3H]DNA and 4 different concentrations of recBC DNase (Fig.  8A). Inhibition by recA protein was most evident at the lower recBC DNase concentrations and was weak at the highest DNase concentration. In no case was inhibition complete. When ATPyS was included in the reaction mixtures, inhibition was strongly enhanced recA protein effectively inhibited the DNase even at the highest DNase concentration (Fig. 8B).
As in the case of exonuclease inhibition, we did an experiment to see whether inhibition can be abolished by raising the DNA concentration while keeping the amounts of recA protein and recBC DNase constant. The results (Fig. 9A) are similar to those observed in the inhibition of the exonuclease activity; inhibition of the endonuclease activity was less effec- Reaction mixtures were prepared as described in Fig. 5, except that recBC DNase was omitted and ATPyS was included in all samples. After incubation at 37 "C for 30 min, each 20-11 sample was mixed with 195 p1 of BD buffer (31 mM Tris-HC1, pH 7.5, 6.7 mM MgC12, 1.8 mM dithiothreitol, 88 pg of BSA/ml) and held on ice. Aliquots of 160 p1 were filtered under suction through a nitrocellulose filter (Millipore DAWP, 0.65 pm pore size; soaked in BD buffer at room temperature for at least 30 min and washed with 2 ml of BD buffer just before use) at a flow rate of 1 m1/10 s. The filters were rinsed with I ml of BD buffer, dried, and counted in 5 ml of Econofluor. Total DNA was determined by spotting 40-pl aliquots of each sample on Sartorius nitrocellulose filters (see "Experimental Procedures") and counting the dried filters in Econofluor. Background binding in the absence of recA protein was between 0.4 and 0.7%. ["]DNA heated at 100 "C for 15 min), 0 or 10 nmol of ATPyS, recA protein as indicated. Incubation was at 37 "C for 5 rnin. The samples were chilled a t 0 "C for 5 min, and 2 units of exonuclease I were added in 5 p1 of recBC diluent (see "Experimental Procedures"). Incubation was at 37 "C for 30 min. Acid-soluble DNA was measured as described under "Experimental Procedures." 0, without ATPyS A, with ATPyS.  The stoichiometry of inhibition of the endonuclease activity of recBC DNase. A, the design of this experiment is as described for the analogous experiment on the inhibition of the exonuclease activity (see Fig. 5 ) . The reaction mixtures (20 pl) contained 10 mM Tris-HC1, pH 7.5,1.5 mM dithiothreitol, 1.3 mg of BSA/ ml, 10 mM MgC12, 1 m~ ATP, 0 or 0.5 mM ATP-@, 5, 10, or 20 p~ fd phage ["]DNA, 0 or 0.6 p~ recA protein, 0.03 unit of recBC DNase. Incubation was at 37 "C for 30 rnin. Endonuclease activity was assayed as described under "Experimental Procedures." 0, without ATPyS; 0, with ATPyS. B, the data from A plotted on double log coordinates. tive at the higher concentrations of DNA. By plotting the data of Fig. 9A on double log coordinates and extrapolating the resulting straight lines to zero inhibition ( i e . 100% nuclease activity), we determined that when there is l molecule of recA protein/40 nucleotide residues, recA protein apparently fails to inhibit the endonuclease activity either in the presence or absence of ATPyS (Fig. 9B). Thus, as one might expect, much more recA protein, 20 times more, is required to protect DNA from endonucleolytic attack than from exonucleolytic attack (see above).

RecA Protein Protects DNA from Degradation
The Effect of RecA Protein on the Degradation of Doublestranded DNA-RecA protein, double-stranded DNA, and single-stranded DNA (or oligodeoxynucleotides) form a ter-nary complex which is trapped in a stable form in the presence of ATPyS (Shibata et al., 1979b). To learn whether the double-stranded DNA in a ternary complex is protected from recBC DNase, we incubated double-stranded Mu [3H]DNA with increasing amounts of recA protein in the presence or absence of ATPyS or oligodeoxynucleotides and then added recBC DNase and ATP to initiate digestion. In the absence of ATPyS, nuclease activity was not affected by either recA protein or oligodeoxynucleotides (Fig. 1OA). In the presence of ATPyS and oligodeoxynucleotides, recA protein inhibited the DNase; inhibition was weak when oligodeoxynucleotides were omitted (Fig. 10B). These results suggest that recA protein inhibits recBC DNase by binding to DNA, since both inhibition and the stable binding of recA protein to doublestranded DNA require ATPyS and are stimulated by oligodeoxynucleotides (Shibata et al., 1979b). The following two experiments support this interpretation.
First, if recA protein protectively binds to double-stranded DNA, then other exonucleases should be inhibited by recA protein in the presence of ATPyS, and inhibition should be enhanced by single-stranded DNA. This was found to be the case in an experiment in which h exonuclease was substituted for recBC DNase (Fig. 10, C and D). Since it is not likely that recA protein specifically interacts with both recBC DNase and h exonuclease, this result supports the idea that inhibition is caused by the binding of recA protein to the DNA substrate.
Secondly, if a direct recA protein-recBC DNase interaction were involved, then the amount of recA protein necessary for Incubation was at 37 "C for 10 min. The samples were chilled a t 0 "C for 3 min, and 60 nmol of ATP in 12 p1 of water and 0.14 unit of recBC DNase in 15 pl of recBC diluent was added. Incubation was at 37 "C for 30 min. Acid-soluble DNA was determined as described under "Experimental Procedures." A, without ATPyS; B, with ATP@; 0, without oligodeoxynucleotides; A, with oligodeoxynucleotides. C and 0, inhibition of X exonuclease by recA protein. Reaction mixtures (15 pl) contained 0.25 pmol of Tris-HCI, pH 7.5, 25 nmol dithiothreitol, 20 pg of BSA, 0.2 pmol of MgC12,20 nmol of ATP, 0 or 10 nmol of ATPyS, 0 or 50 pmol of unlabeled single-stranded DNA fragments (+X174& phage DNA heated at 100 "C for 10 min in 10 mM Tris-HCI, pH 7.5, 0.1 mM EDTA), 100 pmol of Mu ["HIDNA, 0, 24, or 48 pmol of recA protein. After incubation at 37 "C for 10 min, the samples were chilled at 0 "C for 3 min, and 0.1 unit of X exonuclease was added in a volume of 5p1 of recBC diluent. Incubation was continued at 37 "C for 30 min. Acid-soluble Mu DNA was measured as described under "Experimental Procedures." A, without ATPyS; B, with ATPyS; 0, without single-stranded fragments; A, with single-stranded fragments.  (Fig. 11). Thus, a 7-fold change in recBC DNase concentration caused little or no change in the recA protein concentration required for 50% inhibition. This result argues against an inhibitory interaction between recA protein and recBC DNase.

RecA
Double-stranded DNA molecules with single-stranded tails are intermediates in the degradation of double-stranded DNA by recBC DNase (MacKay and Linn, 1974). This raises the possibility that recA protein inhibits recBC DNase by binding to single-stranded tails, rather than to double-stranded regions. To investigate this possibility, we prepared doublestranded Mu DNA with single-stranded tails roughly 1000 nucleotides long (tailed Mu DNA) by digesting native Mu [3H]DNA with X exonuclease (see "Experimental Procedures''). We incubated increasing amounts of recA protein with recBC DNase and either native or tailed Mu DNA in the presence or absence of ATPyS. As already shown above (Fig.   lo), protection of native Mu DNA was weak and was dependent on ATPyS (Fig. 12A). In contrast, tailed Mu DNA was efficiently protected in both the presence and absence of ATPyS (Fig. 12B). This observation is consistent with the ability of recA protein to protect single-stranded DNA in the absence of ATPyS (see Figs. 4 and 8). Because the protection of native Mu DNA requires ATPyS, we conclude that protection is not related to the creation of single-stranded DNA by recBC DNase.
Special Substrates: Gapped DNA and D-loops-Gaps are involved in postreplication repair (Rupp and Howard-Flanders, 1968) and are utilized by recA protein in the homologous pairing of DNA molecules Cunningham et al., 1980). RecBC DNase degrades gapped DNA (Karu et al., 1973) between single-stranded and double-stranded DNA (Prell and Wackernagel, 1980). Since recBC DNase does not attack circular double-stranded DNA (Karu et al., 1973), reckless chromosome degradation in a recA-mutant may be initiated at a gap. Because of these observations, we were interested in studying the interaction between recA protein and recBC DNase on gapped DNA.
Since recA protein appears to catalyze strand transfer from a gap (Cunningham et al., 1980;DasGupta et al., 1980DasGupta et al., , 1981 and since recBC DNase has a DNA unwinding activity in addition to its nuclease activities (Rosamond et al., 1979, Taylor and, we prepared a gapped substrate in which it would be possible to detect the enlargement of gaps. The gapped substrate used in the following experiments was made by annealing circular 4X174am3 [?H]DNA with denatured restriction fragments of @X174arn3 form I [32P]DNA (see "Experimental Procedures"). The restriction fragments were prepared by digestion with restriction endonuclease Hue 111, which cleaves 4X174am3 form I DNA into 11 fragments, ranging in size from 72 to 1353 base pairs (Fuchs et al., 1978). The resulting [3H, 32P]DNA substrate (gDNA) was separated from excess [32P]DNA fragments by sedimentation (Fig. 1). We calculated that the average gDNA molecule was doublestranded along 72% of its length (see "Experimental Procedures''). The ratio of '"P to 3H cpm was 1.5:l at the time that the experiment of Fig. 13 was done (see below). As described under "Experimental Procedures," analysis of gDNA by gel electrophoresis revealed a band of circular gDNA (80% of the total) and a band of linear gDNA (20% of the total). In the following experiments, the endonucleolytic cleavage of gDNA was measured by electrophoretic separation of circular gDNA from linear gDNA and smaller degradation products. The band of circular gDNA was excised and the amounts of "H and 'lP were determined. The amount of circular gDNA present before enzymatic cleavage was determined from a were added to reaction mixtures containing fixed amounts of recBC DNase and gapped circular rJH '''PlDNA (see "Experimental Procedures"). Acid-soluble DNA anh the amount of circular DNA were measured as described below. The 50-p1 reaction mixtures contained 15 mM Tris-HC1, pH 7.5, 1.5 mM dithiothreitol, 1.2 mg of BSA/ml, 10 mM MgC12, 1 mM ATP, 10 p~ gDNA, recA protein as indicated, 14.5 units of recBC DNase/ml. Incubation was at 37 "C for 30 min. A IOpl aliquot was removed for the determination of acid-soluble DNA as described under "Experimental Procedures." For measuring the endonucleolytic cleavage of gDNA, we mixed 40 p1 of each sample with 5 p1 of 2% sodium lauryl sulfate, 125 mM EDTA. Incubation was a t 37 "C for 5 min. Five p1 of proteinase K (2.75 mg/ml) were added and incubation was continued at 37 "C for 15 min. The samples were analyzed by electrophoresis in a 1.2% agarose gel at 8.8 V/cm for 3.5 h. The bands that contained circular gDNA (see Fig. 2) were excised, dissolved in 0.1 N HC1 a t 100 "C, and counted in Triton fluor. The amount of circular gDNA was calculated by comparing the amounts of "H and in the band of circular gDNA to the corresponding amounts of these isotopes in a control sample, from which both recBC DNase and recA protein were omitted. The amount of circular gDNA in the control was defined as 100% Percentage of ,'H (0) and "P (A) in circular gDNA and acid-soluble "H (0) and R2P (A) in the complete reaction mixture are shown. control from which recBC DNase was omitted. Exonuclease activity was assayed by the determination of acid-soluble nucleotides.
T o study the effect of recA protein on the degradation of gDNA by recBC DNase, we incubated various amounts of recA protein with gDNA and recBC DNase (Fig. 13). In the absence of recA protein, only 43% of the "H and 44% of the '"P which were originally present in the circular gDNA remained in the band of circular gDNA after cleavage by recBC DNase.
In the same sample, 29% of the 'H and 18% of the :'2P were made acid-soluble. RecA protein inhibited the endonuclease completely at a recA protein concentration of 5 IJM (corresponding to 1.8 recA monomers/nucleotide residue of singlestranded DNA). Inhibition of the exonuclease was incomplete; 10% of both 'H and 32P nucleotides were made acid-soluble in the presence of 5 IJM recA protein. Since the gDNA preparation was free of acid-soluble nucleotides (see Fig. 2) and since the circular gDNA was completely protected, the acid-soluble DNA produced in the presence of recA protein was probably a consequence of the degradation of linear gDNA.
If recA protein and recBC DNase were able to enlarge gaps, the ratio of :'2P to 'H in circular gDNA would decrease, relative to the ratio in untreated gDNA. In the presence of recBC DNase alone or in combination with various amounts of recA protein, the 'HH:"~P ratio was not significantly affected (Fig.  13). In a control, with 5 IJM recA protein but no recBC DNase, the ratio of '12P to 'H was not affected, relative to that in untreated gDNA. From the observed variation of the ratios in different samples (Fig. 13), we estimate that the ratio of '"P to 'H would have to change by more than 10% to be significant. (The basis for this variation probably lies in our inability to completely resolve circular gDNA from linear gDNA by gel electrophoresis (see Fig. 3.) Since GX174am3 form I DNA is 5375 base pairs long (Sanger et al., 1977), a 10% error implies that more than 540 bases would have to be removed from each gDNA molecule to be detected by our method. Within this limit, we were unable to detect gap enlargement by recA protein or recBC DNase alone or in combination.
In vitro studies have shown that recA protein catalyzes the formation of D-loops (Shibata et al., 1979a) and that in the absence of recA protein recBC DNase endonucleolytically cleaves the D-loop structure (Wiegand et al., 1977). Since both recA protein (this work) and the E. coli single-stranded DNA binding protein (MacKay and Linn, 1976) have been shown to inhibit the endonuclease activity of recBC DNase, we were interested in the effect of these two proteins on the cleavage of D-loops by recBC DNase.
D-loops were prepared by incubating single-stranded fragments of @X174am3 DNA with GX174am3 form I ["HIDNA at 75 "C for 30 min. D-loops were separated from excess fragments by sedimentation through a neutral sucrose gradient. Unbroken DNA (closed duplex, or cdDNA) (Bauer, 1978) constituted 95% of the reisolated ["HIDNA, and 81% of the ["]DNA contained D-loops (see "Experimental Procedures"). This indicates that most molecules of closed duplex DNA contained D-loops. By the time the following experiments had been completed, the cDNA content had fallen to RecA protein and SSB inhibit the endonucleolytic cleavage of Dloops by recBC DNase Purified D-loops were incubated with various combinations of recA protein, SSB, and recBC DNase. D-loop content was determined by an assay which measures the binding of D-loops to a nitrocellulose filter (Beattie et al., 1977). Endonucleolytic cleavage of the DNA was measured by alkaline sucrose gradient sedimentation. The reaction mixtures (40 pl) contained 12.5 mM Tris-HC1, pH 7.5, 2.5 mM potassium phosphate, pH 6.8, 1.2 mM dithiothreitol, 1 mg of BSA/ml, 10 mM MgClz, 1 mM ATP, 0 or 0.5 mM ATP& 5 p~ purified D-loops made from GX174am3 form I ["HIDNA (see "Experimental Procedures"), 0 or 2 p~ recA protein, 0 or 0.6 p~ SSB, 0 or 25 units of recBC DNase/ml. Incubation was at 37 "C for 30 min. The reaction was terminated by the addition of 20 pl of 250 m EDTA. Duplicate 10-pl aliquots were mixed with 10 p1 of 250 mM EDTA and were held on ice for less than 1 h before the D-loop content was measured (see below). The remaining sample was mixed with 100 pl of 25 mM EDTA and 60 p1 of 1 N NaOH, layered on a 5-ml 5-2076 alkaline sucrose gradient (in 0.3 N NaOH, 0.7 M NaCl, 10 nm EDTA), and centrifuged at 50,000 rpm for 70 min at 20 "C in a Beckman SW50.1 rotor. Under these conditions, closed duplex DNA sedimented about two-thirds of the way into the gradient, whereas nicked circular DNA sedimented one-fourth of the way into the gradient (Wiegand et al., 1977). Dloops were assayed as described under "Experimental Procedures," except that each 20-pl sample was incubated at 17 "C for 5 min in the nresence of 1 ul of 10% Sarkosvl prior to the addition of NaCl/Na-"~ Litrate. The 4i "C incubation step was also omitted. 66% and the D-loop content had dropped to 49%. Using alkaline sucrose gradient sedimentation, we measured the cleavage of D-loops by the conversion of cdDNA to nicked DNA and smaller degradation products. D-loop content was determined by an assay in which single-stranded DNA ( i e . D-loops and partially degraded DNA) is retained by a nitrocellulose filter.
In the presence of recA protein, we found that the cleavage of D-loops by recBC DNase was strongly inhibited (Williams, 1981). However, since D-loops are dissociated by recA protein (Shibata et al., 1981b), it is not possible to say whether inhibition is due to the protection of the D-loop structure or to the dissociation of D-loops. However, in the presence of ATPyS, the dissociation of D-loops by recA protein is blocked. We therefore studied the effect of recA protein on the cleavage of D-loops in the presence of ATPyS (Table I, Experiment 1). Untreated DNA contained 51% D-loops and 66% cdDNA. RecA protein alone neither dissociated nor cleaved D-loops. In the presence of both ATPyS and ATP, recBC DNase cleaved D-loops; the amount of cdDNA decreased from 66 to 16% after digestion with the DNase. In the presence of recA protein, however, recBC DNase did not cleave D-loops. We conclude that recA protein inhibits the cleavage of D-loops by recBC DNase in the presence of ATPyS.
To study the effect of the E. coli single-stranded DNA binding protein on D-loop cleavage, we incubated SSB and D-loops in the presence or absence of recBC DNase (Table I, Experiment 2). A control showed that the D-loop preparation contained 66% cdDNA and 51% D-loops (Table I, Experiment 1, Line 1). SSB alone had no effect on the amount of cdDNA or D-loops. In the presence of SSB, recBC DNase did not cleave D-loops by a detectable amount; the cdDNA content was 66% in the untreated D-loops and 62% after treatment with recBC DNase in the presence of SSB. We conclude that SSB does not dissociate D-loops, and that SSB protects Dloops from cleavage by recBC DNase.
In a control (Table I, Experiment 3), +X174an3 form I DNA was not cleaved by recBC DNase, showing that the cleavage of cdDNA in a D-loop preparation is due to the cleavage of D-loops by recBC DNase.

DISCUSSION
We have shown that recA protein inhibits the degradation of single-stranded DNA by the exonuclease and endonuclease activities of recBC DNase. The extent of inhibition depends on the relative concentrations of DNA, recA protein, and recBC DNase. For a given amount of recA protein, inhibition was favored at low concentrations of both recBC DNase (Figs. 4B and 7A) and single-stranded DNA (Figs. 5 and 10). Complete inhibition was not observed with either single-stranded DNA fragments or fd phage DNA as the substrate. However, recA protein completely inhibited the endonucleolytic cleavage of gapped DNA (Fig. 13), in which the concentration of single-stranded DNA was lower than in the two substrates mentioned above. These results indicate that, under the proper conditions, recA protein might protect DNA from degradation by recBC DNase in uiuo, as suggested by the experiments of Satta et al. (1979).
The experiments reported here support the view that by binding to the DNA recA protein protects it from degradation by recBC DNase: 1) at fixed concentrations of recA protein and recBC DNase, inhibition is abolished as the DNA concentration increases (Figs. 5 and 9). 2) A greater amount of recA protein is needed to inhibit the endonuclease activity than the exonuclease activity of recBC DNase. The ratio of recA protein to nucleotide residues below which inhibition was not apparent was 1:40 for the endonuclease and 1:800 for the exonuclease (Figs. 5 and 9). This is consistent with the binding of recA protein to DNA since it would be necessary to cover a large proportion of a DNA molecule to prevent endonucleolytic cleavage, whereas only a few molecules of recA protein bound to the DNA would suffice to block the progress of an exonuclease. 3) ATPyS is required for the protection of double-stranded DNA but not for the protection of singlestranded DNA. This is consistent with the observation that recA protein binds more tightly to single-stranded DNA than to double-stranded DNA (Gudas and Pardee, 1975;Shibata et al., 1979b). 4) ATPyS enhances both the protection of singlestranded DNA (Figs. 4A and 8B) and the binding of recA protein to single-stranded DNA (Shibata et al., 1979b). 5) ATPyS enhances the inhibition of exonuclease I activity on single-stranded DNA (Fig. 7), and ATPyS is required for the inhibition of both recBC DNase and A exonuclease activities on double-stranded DNA (Fig. 10). Thus, the inhibitory effect of recA protein and the enhancement by ATPyS are not specific for recBC DNase. The simplest explanation for this is that recA protein inhibits nucleases by binding to the DNA. 6) Large amonts of recA protein are required for inhibition. For example, 0.5 IJM recA protein was required to half-inhibit the degradation of linear single-stranded DNA by recBC DNase a t a concentration of 8 units/ml (Fig. 4). Assuming that the DNase was 100% pure and given a specific activity of 14,700 units/mg (see "Experimental Procedures"), we can calculate that the concentration of nuclease molecules (Mr = 270,000) was a t most 0.002 PM. In this case, half-inhibition required at least 250 molecules of recA protein/molecule of recBC DNase. If an interaction occurs between recA protein and recBC DNase, it is a weak interaction. On the other hand, the quantities of recA protein required for inhibition are similar to the amount of DNA; half-inhibition occurred at 0.5 p~ recA protein, which corresponds to 1 recA monomer/lO DNA nucleotide residues. West et al. (1980) have estimated that single-stranded DNA is saturated with recA protein when there is 1 recA monomer/4 nucleotides. While these observations indicate that recA protein protects DNA by binding to it, they do not rule out the possibility of a direct interaction between recA protein and recBC DNase.
The protection of double-stranded Mu DNA by recA protein was not significant except in the presence of both ATPyS and oligodeoxynucleotides (Fig. 10). Although this result indicates that recA protein may not protect double-stranded DNA under physiological conditions (see below), it supports the concept of a ternary complex of recA protein, singlestranded DNA, and double-stranded DNA (Shibata et al., 1979b).
Since both recA protein and recBC DNase are able to promote the unwinding of DNA (Cunningham et al., 1980;DasGupta et al., 1980;Rosamond et al., 1979), we looked for the enlargement of gaps in the presence of these enzymes. We estimated that gaps would have to be enlarged by more than 500 to 600 bases/substrate molecule to be detected by our assay. Within this limit, we were unable to detect the extension of gaps in circular gapped DNA by recA protein, recBC DNase, or by a combination of these two enzymes.
If D-loops or similar structures are intermediates in recombination (Shibata et al., 1979a;Cunningham et al., 1980;DasGupta et al., 1980), they must somehow be cleaved to produce the final recombinant molecules. The present in uitro experiments show that SSB alone protects D-loops from cleavage by recBC DNase. Because recA protein in uitro can dissociate D-loops made from single-stranded fragments plus superhelical DNA (Shibata et al., 1981b), we could only study the protection of D-loops by recA protein in the presence of ATPyS. Thus, further experiments are required to get some RecA Protein Protects DNA from Degradation insight into the enzymological basis for resolving intermediates formed in recombination.
Unless there is a functional analog of ATPyS in uiuo, we can conclude that recA protein protects single-stranded DNA but not double-stranded DNA from nucleolytic degradation. Thus, the protective function of recA protein probably acts to prevent the occurrence of double-stranded breaks in the chromosome, possibly by protecting gaps from endonucleolytic cleavage (Fig. 13). Once the exonuclease activity of recBC DNase has gained access to chromosomal DNA, recA protein may be unable to prevent further degradation.