Vol. 174, No. 23
JOURNAL OF BACTERIOLOGY, Dec. 1992, p. 7705-7710
0021-9193/92/237705-06$02.00/0 Copyright X 1992, American Society for Microbiology
Evidence for ATP Binding and Double-Stranded DNA Binding by Escherichia coli RecF Protein MURTY V. V. S. MADIRAJU* AND ALVIN J. CLARK Department of Molecular and Cell Biology, 401 Barker Hall, University of California at Berkeley, Berkeley, California 94720 Received 14 July 1992/Accepted 28 September 1992
RecF protein is one of the important proteins involved in DNA recombination and repair. RecF protein has been shown to bind single-stranded DNA (ssDNA) in the absence of ATP (T. J. Griffin IV and R. D. Kolodner, J. Bacteriol. 172:6291-6299, 1990; M. V. V. S. Madiraju and A. J. Clark, Nucleic Acids Res. 19:6295-6300, 1991). In the present study, using 8-azido-ATP, a photo-affinity analog of ATP, we show that RecF protein binds ATP and that the binding is specific in the presence of DNA. 8-Azido-ATP photo-cross-linking is stimulated in the presence of DNA (both ssDNA and double-stranded DNA [dsDNAJ), suggesting that DNA enhances the affinity of RecF protein for ATP. These data suggest that RecF protein possesses independent ATP- and DNA-binding sites. Further, we find that stable RecF protein-dsDNA complexes are obtained in the presence of ATP or ATP-'y-S [adenosine-5'-O-(3-thio-triphosphate)J. No other nucleoside triphosphates served as necessary cofactors for dsDNA binding, indicating that RecF is an ATP-dependent dsDNA-binding protein. Since a mutation in a putative phosphate-binding motif of RecF protein results in a recF mutant phenotype (S. J. Sandler, B. Chackerian, J. T. Li, and A. J. Clark, Nucleic Acids Res. 20:839-845, 1992), we suggest on the basis of our data that the interactions of RecF protein with ATP, with dsDNA, or with both are physiologically important for understanding RecF protein function in vivo.
otide. Recently, Sandler et al. (15) made a conservative lysine-to-arginine change in this consensus sequence and found that cells containing this mutation showed UV sensitivity. Their study suggests that the normal ability of RecF protein to interact with phosphate-containing cofactor in vivo is impaired in cells containing this conservative change in the RecF protein. ATP and or other nucleoside triphosphates (NTPs) and DNA are some of the phosphate-containing cofactors that might bind to RecF protein. We have undertaken this study to identify the likely cofactors interacting with RecF protein. Using 8-azido-ATP, a photo-affinity analog of ATP, we asked two important questions: (i) does RecF bind ATP and other NTPs, and (ii) if it does, what is the relevance of this ATP or NTP binding? The implications of these results for understanding RecF protein function in vivo are presented.
RecF protein is one of the important proteins involved in DNA recombination and repair. Mutations in the recF gene in a wild-type background cause a delay in induction of the SOS repair response, an inducible DNA repair process, and deficiency in carrying out recombinational repair (see reference 3 for a recent review). A critical step in inducing the SOS response process in vivo is the cleavage of LexA repressor protein in the presence of RecA protein and a cofactor presumed to be single-stranded DNA (ssDNA) (see reference 9 for a review). Recent genetic data clearly indicate that the LexA repressor cleavage reaction is delayed in recF mutants (16, 17). RecA protein has been shown to cleave LexA repressor efficiently in vitro at physiological conditions in the presence of both ATP and ssDNA (9). RecF protein apparently is not needed in this in vitro reaction. Thus, in vivo RecF protein must be involved either in generating the ssDNA needed for LexA repressor cleavage by RecA protein or in allowing the optimal binding of RecA protein to ssDNA. It is also plausible that RecF protein plays a similar role in recombinational repair reac-
MATERIALS AND METHODS Chemicals and buffers. Radiolabeled 32P-,y-8-azido-ATP was obtained from ICN Pharmaceuticals. Nonradioactive 8-azido ATP, ATP, ATP--y-S [adenosine-5'-O-(3-thiotriphosphate)], lysozyme, and trypsin were obtained from Sigma Chemical Co. GTP, UTP, CTP, and TTP were obtained from United States Biochemicals. The enhanced chemiluminescence (ECL) kit was from Amersham. The concentration of 8-azido ATP was determined by using a value of e280 = 13.3 x 103 M-1 cm-1. Proteins and DNA substrates. RecF protein used in the present study was purified essentially as described by Madiraju and Clark (10) with the following modifications. The RecF protein preparation obtained after successive fractionations on Phosphocellulose P-11, PBE-94, and Ultrogel ACA54 resins was additionally fractionated with an ssDNA cellulose column preequilibrated with a buffer containing 20 mM Tris-HCl, 0.1 mM dithiothreitol (DTT), 0.1 mM EDTA,
tions (3). Purified RecF protein has been shown to bind ssDNA in vitro (6, 10). Although this interaction of RecF protein might be physiologically important, no robust models to correlate this in vitro ssDNA-binding property with LexA protein cleavage and recombinational repair reactions have emerged. Clearly, more-detailed biochemical characterization of RecF protein is warranted for understanding its function in vivo. RecF protein has the sequence GxxGxGKT (5, 15), a consensus sequence found in many ATP-binding proteins, ATPases, and GTPases (13, 18, 26). This sequence is associated with the binding of the phosphate moiety of a nucle*
Corresponding author. 7705
7706
MADIRAJU AND CLARK
20% glycerol, and 50 mM NaCl (final concentrations). After the protein was added, the column was washed with 2 column volumes of the buffer used for preequilibration. Bound protein was eluted in a linear gradient containing 50 to 500 mM NaCl in the same buffer. Peak fractions obtained were pooled (12 ml), concentrated (to 4 ml) by using a Centricon filtration device, and dialyzed against buffer containing 50% glycerol. The dialyzed protein was subsequently used in all these assays. This purification step resulted in the removal of a contaminating protein with a molecular mass of 44,000 Da. Though this protein preparation is more homogeneous than our previous preparation (10), removal of this protein does not appear to affect any of the properties investigated in the present study (data not shown). RecA protein was purified as described elsewhere (11). 4X174 ssDNA and its double-stranded DNA (dsDNA) were prepared as described elsewhere (11). Concentrations of DNA are given in moles of nucleotide residues per liter. Photo-cross-linking reactions. Unless otherwise mentioned, all photo (UV)-cross-linking experiments were carried out at room temperature in a buffer containing 20 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 30 mM KCI, and 0.05 mM P-mercaptoethanol. Reaction mixtures with a final volume of 40 ,ul containing RecF protein (0.5 ,uM) and 8-azido-ATP in the reaction buffer just described were incubated for 2 min at 37°C. At the end of the incubation period, samples were spotted on Parafilm and exposed to 1 min of UV irradiation under an 8-W germicidal lamp (254 nm) at a distance of 7.4 cm from the reaction mixtures. This exposure time gives a UV dose of 288 J/m2. Following the irradiation period, DTT was added to a final concentration of 10 mM, and spots on the Parafilm were removed to Eppendorf tubes. After the addition of sodium dodecyl sulfate (SDS)-Laemmli buffer, samples were boiled for 3 min and analyzed by electrophoresis in a 10% SDS-polyacrylamide gel. Following electrophoresis, polyacrylamide gels were soaked in 10% isopropanol-5% acetic acid for 20 min, rinsed with several changes of double-distilled water, dried, and exposed to X-ray film. Where appropriate, autoradiograms were scanned for quantitative measurements with a Hoefer densitometer. The final concentration of 3-mercaptoethanol used in the reaction samples seems to affect the amount of 8-azido-ATP that cross-links to RecF protein. Kowalczykowski (8) showed that nonspecific photo-cross-linking of 8-azido-ATP to RecA protein is reduced in the presence of ,B-mercaptoethanol. We also found that under our reaction conditions in the absence of DNA, use of as little as 0.005 mM 3-mercaptoethanol reduced covalent photo-cross-linking by approximately 70%. Higher concentrations (0.01 to 0.05 mM) of 3-mercaptoethanol reduced the extent of photo-cross-linking by 90% (data not shown). By contrast, in the presence of DNA, only 10% of the total cross-linking signal was lost at the highest concentration of 3-mercaptoethanol (0.05 mM), indicating that photo-cross-linking in the presence of DNA is about 10 times more specific than in its absence. Hence, unless mentioned otherwise, all experiments were carried out in the presence of 0.05 mM ,B-mercaptoethanol. The photo-cross-linking signal in the presence of DNA seems to be specific at least up to a 40 ,uM 8-azido-ATP concentration (data not shown). Agarose gel assay for DNA binding. An agarose gel assay for detecting RecF-dsDNA complexes was used. RecF protein as indicated was incubated for 10 min at 37°C in 20 mM Tris-HCl (pH 7.5)-0.1 mM DT`1T-0.1 mM EDTA-50 mM NaCl-3.0 p,M linear dsDNA. At the end of the incubation period, S ,ul of 0.5 M EDTA was added, and then a gel
J. BACTERIOL.
loading buffer containing 50% glycerol and 0.02% bromophenol blue was added. Protein-DNA complexes were separated from free DNA by running samples at room temperature on 0.8% agarose gels at 4 V/cm per gel in TAE buffer containing 20 mM Tris-HCl, 4 mM sodium acetate, and 0.1 mM EDTA. Following electrophoresis, gels were stained with ethidium bromide and destained for the appropriate time, and proteinDNA complexes were visualized. We did not detect any RecF-dsDNA complexes with TBE (12) buffer. In some experiments, agarose gels were soaked for 30 min in electrophoretic transfer buffer containing 0.01% SDS. Protein samples were then electrophoretically transferred to nitrocellulose filters and were probed with antibodies raised against pure RecF protein by using Amersham's ECL detection system. RESULTS 8-Azido ATP can be photo-cross-linked covalently to RecF protein. A method that has been generally used to study the interactions of a protein with ATP is to photo (UV)-crosslink covalently a photo-affinity analog of ATP, such as 8-azido-ATP, to the protein in question (4). Since RecF protein has a consensus sequence coding for a putative phosphate-binding loop (5, 15), we asked whether 32p__Y labeled 8-azido-ATP could be used as a probe for studying the interaction of ATP with RecF protein. When a buffer containing RecF protein and 8-azido-ATP was UV irradiated, a radiolabeled protein band corresponding to a RecF protein with a molecular mass of 40,000 Da was seen on polyacrylamide gels (data not shown). No cross-linking was noted in the absence of UV irradiation (data not shown). Heat-denatured RecF did not serve as a substrate, indicating that a native conformation of protein is needed for such photo-cross-linking (data not shown). In addition, trypsin and lysozyme, which neither have the putative phosphatebinding consensus sequence nor bind ATP, were not photocross-linked under our experimental conditions (data not shown). Furthermore, RecA protein, which has the consensus sequence and does bind ATP (7), was photo-cross-linked under our reaction conditions, confirming the observations of Knight and McEntee (7) and Kowalczykowski (8; data not
shown). DNA stimulates 8-azido-ATP cross-linking to RecF protein. RecF protein has been shown to bind ssDNA but not dsDNA, suggesting that RecF protein may have an ssDNAbinding site (6, 10). It is conceivable that the phosphate moiety of the DNA backbone interacts with the phosphatebinding-loop motif of the RecF protein sequence. If so, the 8-azido-ATP might be interacting with a putative ssDNAbinding site in RecF protein. In order to test whether 8-azido-ATP is cross-linking to an ssDNA-binding site in RecF protein or to a site(s) with which ATP normally interacts in RecF protein, i.e., an ATP-binding site, we carried out photo-cross-linking studies in the presence of different amounts of ssDNA. If 8-azido-ATP were to interact with a putative ssDNA-binding site, we would expect inhibition of (interference with) photo-cross-linking in the presence of ssDNA. Because our earlier experiments (10) and those of Griffin and Kolodner (6) indicated that RecF protein does not bind dsDNA, we carried out photo-cross-linking in the presence of dsDNA, because we expected no inhibition of photo-cross-linking. Since higher concentrations of DNA (UV-absorbing material) used in the reactions might quench UV light, thereby decreasing the photo-cross-linking signal, we used adenosine as a control for UV-absorbing material.
PROPERTIES OF RecF PROTEIN
VOL. 174, 1992
Adenosine .0
n
N
0 C)000 to
0L___
ssDNA
7707
dsDNA 0
0
0C 0CD00 0 N Li-U NiU°°0C 0
0
.
0
8
60
FIG. 1. Photo-cross-linking of 8-azido-ATP in the presence of DNA. RecF protein (0.5 ,uM) was incubated for 2 min at 37°C with different amounts of DNA (ssDNA or dsDNA) or adenosine in a reaction buffer containing 20 mM Tris-HCl, 10 mM MgCl2, and 30 mM KCI. P-Mercaptoethanol was added to a final concentration of 0.05 mM. At the end of incubation, samples were UV irradiated for 1 min as described in Materials and Methods. The photo-crosslinking reaction was stopped by adding DTT to a final concentration of 10 mM. Samples were separated on SDS-polyacrylamide gels, and the cross-linked proteins were identified after autoradiography. N-base, UV absorbing material (DNA or adenosine).
40
o
20 0
10
20
30
40
8-Azido-ATP Conc. (microM) Photo-cross-linking experiments were carried out with 0.5 ,uM RecF protein and 5.0 ,M 8-azido-ATP. The results of the experiments are shown in Fig. 1. Two distinct observations can be made. First, in the presence of either ssDNA or dsDNA, 8-azido-ATP is not prevented from covalent cross-linking to RecF protein. This indicates that the site with which ATP is interacting in RecF protein is distinctly different from the site(s) with which DNA interacts. Second, both ssDNA and dsDNA stimulated the 8-azido-ATP cross-linking (compare lanes with DNA and those with adenosine in Fig. 1). Since dsDNA also stimulated the photo-cross-linking reaction, we further interpreted from these data that RecF protein binds dsDNA in the presence of ATP. Our data given below are consistent with this interpretation. The experiments described above were carried out at 0.5 ,uM RecF and 5.0 ,uM 8-azido-ATP. Under these conditions, 10 molecules of 8-azido-ATP were present for every molecule of RecF protein. If the interpretation given above, i.e., that binding of DNA increases RecF protein's ability to bind 8-azido-ATP, thereby resulting in the enhanced cross-linking signal, is correct, then we expect enhanced cross-linking in the presence of DNA even at other ratios of 8-azido-ATP to RecF protein. To test this, we carried out 8-azido-ATP cross-linking experiments in the presence of a fixed concentration of DNA but at different concentrations of 8-azidoATP. No adenosine control reaction was used in this experiment because no UV quenching at this concentration of DNA (i.e., 10 ,uM) was noted. For comparison, experiments were also done in the absence of DNA. Reaction samples after analysis on SDS-polyacrylamide gels were autoradiographed and scanned with a Hoefer densitometer. Peak intensities obtained at saturation were set at 100% for both DNA and no-DNA samples. The fractions of total saturation at different azido-ATP concentrations were then determined as shown in Fig. 2. Our results show that as the concentration of 8-azido-ATP increases in the absence of DNA, the fraction of total azido-ATP bound increases sigmoidally. In contrast, a linear increase followed by saturation of photo-cross-linking was noted in the presence of DNA (Fig. 2). This suggests that photo-cross-linking in the absence of DNA is cooperative, while cross-linking in the presence of DNA is not cooperative. Consistent with this interpretation, we noted that the
FIG. 2. Photo-cross-linking in the presence of different concentrations of 8-azido-ATP. RecF protein (0.5 p,M) was incubated with different amounts of 8-azido-ATP in the presence (*) and the absence (0) of ssDNA (5.0 F.M). All subsequent reaction conditions are as described in the legend to Fig. 1. Autoradiograms obtained were scanned with a Hoefer densitometer. From the peak values obtained, the fraction of total protein saturated was calculated.
absolute (i.e., not normalized) amount of photo-cross-linking at saturation is higher for reactions containing no DNA than for those containing DNA (data not shown). This suggests that in the absence of DNA, photo-cross-linking to both a DNA- and an ATP-binding site might be occurring, while in the presence of DNA, photo-cross-linking only to the ATPbinding site may occur. This suggestion could be tested by labeling and isolating the specific peptide(s) cross-linked with 8-azido-ATP for sequence analysis. From our data, it is difficult to determine the stoichiometry of ATP binding to RecF protein. We do not know what proportion of RecF monomers and/or 8-azido-ATP molecules is active in our experiments. Assuming that every RecF monomer and 8-azido-ATP molecule is active, then one can accurately determine the number of 8-azido-ATP molecules bound per RecF monomer (i.e., stoichiometry) at any given time both in the presence and in the absence of DNA. Our future experiments will address this problem. ATP competes with 8-azido-ATP cross-linking to RecF. An important criterion for determining whether 8-azido-ATP is photo-cross-linking at a specific site, e.g., an ATP-binding site, in RecF protein, is to ask whether natural analogs such as ATP, GTP, C1JP, and TJP can compete with 8-azido-ATP and thereby inhibit photo-cross-linking. If the photo-crosslinking is at a site which is nonspecific, then none of these natural analogs are expected to compete with 8-azido-ATP for cross-linking to RecF protein. To test this, we carried out photo-cross-linking experiments in the presence of different NTPs. Since the NTP used in the reactions might also inhibit photo-cross-linking by quenching UV light, parallel control reactions with adenosine were also carried out. Our results indicated that only ATP (Fig. 3) competed with 8-azido-ATP for binding to RecF. However, other NTPs such as GTP, CTP, UTP, and T at up to 500 mM did not compete with 8-azido-ATP for binding to RecF protein (data not shown). In Fig. 3, we can clearly see stimulation of photo-cross-
7708
MADIRAJU AND CLARK -ATP
o
0.
*R
No ATP
+ ATP
Adenosine
o
J. BACTERIOL.
ATP-y-S
dsDNA
ssDNA
0 0o°
,*CM
ATP
r9 A 4e 6N;~+ N
r
JOURNAL OF BACTERIOLOGY, Dec. 1992, p. 7705-7710
0021-9193/92/237705-06$02.00/0 Copyright X 1992, American Society for Microbiology
Evidence for ATP Binding and Double-Stranded DNA Binding by Escherichia coli RecF Protein MURTY V. V. S. MADIRAJU* AND ALVIN J. CLARK Department of Molecular and Cell Biology, 401 Barker Hall, University of California at Berkeley, Berkeley, California 94720 Received 14 July 1992/Accepted 28 September 1992
RecF protein is one of the important proteins involved in DNA recombination and repair. RecF protein has been shown to bind single-stranded DNA (ssDNA) in the absence of ATP (T. J. Griffin IV and R. D. Kolodner, J. Bacteriol. 172:6291-6299, 1990; M. V. V. S. Madiraju and A. J. Clark, Nucleic Acids Res. 19:6295-6300, 1991). In the present study, using 8-azido-ATP, a photo-affinity analog of ATP, we show that RecF protein binds ATP and that the binding is specific in the presence of DNA. 8-Azido-ATP photo-cross-linking is stimulated in the presence of DNA (both ssDNA and double-stranded DNA [dsDNAJ), suggesting that DNA enhances the affinity of RecF protein for ATP. These data suggest that RecF protein possesses independent ATP- and DNA-binding sites. Further, we find that stable RecF protein-dsDNA complexes are obtained in the presence of ATP or ATP-'y-S [adenosine-5'-O-(3-thio-triphosphate)J. No other nucleoside triphosphates served as necessary cofactors for dsDNA binding, indicating that RecF is an ATP-dependent dsDNA-binding protein. Since a mutation in a putative phosphate-binding motif of RecF protein results in a recF mutant phenotype (S. J. Sandler, B. Chackerian, J. T. Li, and A. J. Clark, Nucleic Acids Res. 20:839-845, 1992), we suggest on the basis of our data that the interactions of RecF protein with ATP, with dsDNA, or with both are physiologically important for understanding RecF protein function in vivo.
otide. Recently, Sandler et al. (15) made a conservative lysine-to-arginine change in this consensus sequence and found that cells containing this mutation showed UV sensitivity. Their study suggests that the normal ability of RecF protein to interact with phosphate-containing cofactor in vivo is impaired in cells containing this conservative change in the RecF protein. ATP and or other nucleoside triphosphates (NTPs) and DNA are some of the phosphate-containing cofactors that might bind to RecF protein. We have undertaken this study to identify the likely cofactors interacting with RecF protein. Using 8-azido-ATP, a photo-affinity analog of ATP, we asked two important questions: (i) does RecF bind ATP and other NTPs, and (ii) if it does, what is the relevance of this ATP or NTP binding? The implications of these results for understanding RecF protein function in vivo are presented.
RecF protein is one of the important proteins involved in DNA recombination and repair. Mutations in the recF gene in a wild-type background cause a delay in induction of the SOS repair response, an inducible DNA repair process, and deficiency in carrying out recombinational repair (see reference 3 for a recent review). A critical step in inducing the SOS response process in vivo is the cleavage of LexA repressor protein in the presence of RecA protein and a cofactor presumed to be single-stranded DNA (ssDNA) (see reference 9 for a review). Recent genetic data clearly indicate that the LexA repressor cleavage reaction is delayed in recF mutants (16, 17). RecA protein has been shown to cleave LexA repressor efficiently in vitro at physiological conditions in the presence of both ATP and ssDNA (9). RecF protein apparently is not needed in this in vitro reaction. Thus, in vivo RecF protein must be involved either in generating the ssDNA needed for LexA repressor cleavage by RecA protein or in allowing the optimal binding of RecA protein to ssDNA. It is also plausible that RecF protein plays a similar role in recombinational repair reac-
MATERIALS AND METHODS Chemicals and buffers. Radiolabeled 32P-,y-8-azido-ATP was obtained from ICN Pharmaceuticals. Nonradioactive 8-azido ATP, ATP, ATP--y-S [adenosine-5'-O-(3-thiotriphosphate)], lysozyme, and trypsin were obtained from Sigma Chemical Co. GTP, UTP, CTP, and TTP were obtained from United States Biochemicals. The enhanced chemiluminescence (ECL) kit was from Amersham. The concentration of 8-azido ATP was determined by using a value of e280 = 13.3 x 103 M-1 cm-1. Proteins and DNA substrates. RecF protein used in the present study was purified essentially as described by Madiraju and Clark (10) with the following modifications. The RecF protein preparation obtained after successive fractionations on Phosphocellulose P-11, PBE-94, and Ultrogel ACA54 resins was additionally fractionated with an ssDNA cellulose column preequilibrated with a buffer containing 20 mM Tris-HCl, 0.1 mM dithiothreitol (DTT), 0.1 mM EDTA,
tions (3). Purified RecF protein has been shown to bind ssDNA in vitro (6, 10). Although this interaction of RecF protein might be physiologically important, no robust models to correlate this in vitro ssDNA-binding property with LexA protein cleavage and recombinational repair reactions have emerged. Clearly, more-detailed biochemical characterization of RecF protein is warranted for understanding its function in vivo. RecF protein has the sequence GxxGxGKT (5, 15), a consensus sequence found in many ATP-binding proteins, ATPases, and GTPases (13, 18, 26). This sequence is associated with the binding of the phosphate moiety of a nucle*
Corresponding author. 7705
7706
MADIRAJU AND CLARK
20% glycerol, and 50 mM NaCl (final concentrations). After the protein was added, the column was washed with 2 column volumes of the buffer used for preequilibration. Bound protein was eluted in a linear gradient containing 50 to 500 mM NaCl in the same buffer. Peak fractions obtained were pooled (12 ml), concentrated (to 4 ml) by using a Centricon filtration device, and dialyzed against buffer containing 50% glycerol. The dialyzed protein was subsequently used in all these assays. This purification step resulted in the removal of a contaminating protein with a molecular mass of 44,000 Da. Though this protein preparation is more homogeneous than our previous preparation (10), removal of this protein does not appear to affect any of the properties investigated in the present study (data not shown). RecA protein was purified as described elsewhere (11). 4X174 ssDNA and its double-stranded DNA (dsDNA) were prepared as described elsewhere (11). Concentrations of DNA are given in moles of nucleotide residues per liter. Photo-cross-linking reactions. Unless otherwise mentioned, all photo (UV)-cross-linking experiments were carried out at room temperature in a buffer containing 20 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 30 mM KCI, and 0.05 mM P-mercaptoethanol. Reaction mixtures with a final volume of 40 ,ul containing RecF protein (0.5 ,uM) and 8-azido-ATP in the reaction buffer just described were incubated for 2 min at 37°C. At the end of the incubation period, samples were spotted on Parafilm and exposed to 1 min of UV irradiation under an 8-W germicidal lamp (254 nm) at a distance of 7.4 cm from the reaction mixtures. This exposure time gives a UV dose of 288 J/m2. Following the irradiation period, DTT was added to a final concentration of 10 mM, and spots on the Parafilm were removed to Eppendorf tubes. After the addition of sodium dodecyl sulfate (SDS)-Laemmli buffer, samples were boiled for 3 min and analyzed by electrophoresis in a 10% SDS-polyacrylamide gel. Following electrophoresis, polyacrylamide gels were soaked in 10% isopropanol-5% acetic acid for 20 min, rinsed with several changes of double-distilled water, dried, and exposed to X-ray film. Where appropriate, autoradiograms were scanned for quantitative measurements with a Hoefer densitometer. The final concentration of 3-mercaptoethanol used in the reaction samples seems to affect the amount of 8-azido-ATP that cross-links to RecF protein. Kowalczykowski (8) showed that nonspecific photo-cross-linking of 8-azido-ATP to RecA protein is reduced in the presence of ,B-mercaptoethanol. We also found that under our reaction conditions in the absence of DNA, use of as little as 0.005 mM 3-mercaptoethanol reduced covalent photo-cross-linking by approximately 70%. Higher concentrations (0.01 to 0.05 mM) of 3-mercaptoethanol reduced the extent of photo-cross-linking by 90% (data not shown). By contrast, in the presence of DNA, only 10% of the total cross-linking signal was lost at the highest concentration of 3-mercaptoethanol (0.05 mM), indicating that photo-cross-linking in the presence of DNA is about 10 times more specific than in its absence. Hence, unless mentioned otherwise, all experiments were carried out in the presence of 0.05 mM ,B-mercaptoethanol. The photo-cross-linking signal in the presence of DNA seems to be specific at least up to a 40 ,uM 8-azido-ATP concentration (data not shown). Agarose gel assay for DNA binding. An agarose gel assay for detecting RecF-dsDNA complexes was used. RecF protein as indicated was incubated for 10 min at 37°C in 20 mM Tris-HCl (pH 7.5)-0.1 mM DT`1T-0.1 mM EDTA-50 mM NaCl-3.0 p,M linear dsDNA. At the end of the incubation period, S ,ul of 0.5 M EDTA was added, and then a gel
J. BACTERIOL.
loading buffer containing 50% glycerol and 0.02% bromophenol blue was added. Protein-DNA complexes were separated from free DNA by running samples at room temperature on 0.8% agarose gels at 4 V/cm per gel in TAE buffer containing 20 mM Tris-HCl, 4 mM sodium acetate, and 0.1 mM EDTA. Following electrophoresis, gels were stained with ethidium bromide and destained for the appropriate time, and proteinDNA complexes were visualized. We did not detect any RecF-dsDNA complexes with TBE (12) buffer. In some experiments, agarose gels were soaked for 30 min in electrophoretic transfer buffer containing 0.01% SDS. Protein samples were then electrophoretically transferred to nitrocellulose filters and were probed with antibodies raised against pure RecF protein by using Amersham's ECL detection system. RESULTS 8-Azido ATP can be photo-cross-linked covalently to RecF protein. A method that has been generally used to study the interactions of a protein with ATP is to photo (UV)-crosslink covalently a photo-affinity analog of ATP, such as 8-azido-ATP, to the protein in question (4). Since RecF protein has a consensus sequence coding for a putative phosphate-binding loop (5, 15), we asked whether 32p__Y labeled 8-azido-ATP could be used as a probe for studying the interaction of ATP with RecF protein. When a buffer containing RecF protein and 8-azido-ATP was UV irradiated, a radiolabeled protein band corresponding to a RecF protein with a molecular mass of 40,000 Da was seen on polyacrylamide gels (data not shown). No cross-linking was noted in the absence of UV irradiation (data not shown). Heat-denatured RecF did not serve as a substrate, indicating that a native conformation of protein is needed for such photo-cross-linking (data not shown). In addition, trypsin and lysozyme, which neither have the putative phosphatebinding consensus sequence nor bind ATP, were not photocross-linked under our experimental conditions (data not shown). Furthermore, RecA protein, which has the consensus sequence and does bind ATP (7), was photo-cross-linked under our reaction conditions, confirming the observations of Knight and McEntee (7) and Kowalczykowski (8; data not
shown). DNA stimulates 8-azido-ATP cross-linking to RecF protein. RecF protein has been shown to bind ssDNA but not dsDNA, suggesting that RecF protein may have an ssDNAbinding site (6, 10). It is conceivable that the phosphate moiety of the DNA backbone interacts with the phosphatebinding-loop motif of the RecF protein sequence. If so, the 8-azido-ATP might be interacting with a putative ssDNAbinding site in RecF protein. In order to test whether 8-azido-ATP is cross-linking to an ssDNA-binding site in RecF protein or to a site(s) with which ATP normally interacts in RecF protein, i.e., an ATP-binding site, we carried out photo-cross-linking studies in the presence of different amounts of ssDNA. If 8-azido-ATP were to interact with a putative ssDNA-binding site, we would expect inhibition of (interference with) photo-cross-linking in the presence of ssDNA. Because our earlier experiments (10) and those of Griffin and Kolodner (6) indicated that RecF protein does not bind dsDNA, we carried out photo-cross-linking in the presence of dsDNA, because we expected no inhibition of photo-cross-linking. Since higher concentrations of DNA (UV-absorbing material) used in the reactions might quench UV light, thereby decreasing the photo-cross-linking signal, we used adenosine as a control for UV-absorbing material.
PROPERTIES OF RecF PROTEIN
VOL. 174, 1992
Adenosine .0
n
N
0 C)000 to
0L___
ssDNA
7707
dsDNA 0
0
0C 0CD00 0 N Li-U NiU°°0C 0
0
.
0
8
60
FIG. 1. Photo-cross-linking of 8-azido-ATP in the presence of DNA. RecF protein (0.5 ,uM) was incubated for 2 min at 37°C with different amounts of DNA (ssDNA or dsDNA) or adenosine in a reaction buffer containing 20 mM Tris-HCl, 10 mM MgCl2, and 30 mM KCI. P-Mercaptoethanol was added to a final concentration of 0.05 mM. At the end of incubation, samples were UV irradiated for 1 min as described in Materials and Methods. The photo-crosslinking reaction was stopped by adding DTT to a final concentration of 10 mM. Samples were separated on SDS-polyacrylamide gels, and the cross-linked proteins were identified after autoradiography. N-base, UV absorbing material (DNA or adenosine).
40
o
20 0
10
20
30
40
8-Azido-ATP Conc. (microM) Photo-cross-linking experiments were carried out with 0.5 ,uM RecF protein and 5.0 ,M 8-azido-ATP. The results of the experiments are shown in Fig. 1. Two distinct observations can be made. First, in the presence of either ssDNA or dsDNA, 8-azido-ATP is not prevented from covalent cross-linking to RecF protein. This indicates that the site with which ATP is interacting in RecF protein is distinctly different from the site(s) with which DNA interacts. Second, both ssDNA and dsDNA stimulated the 8-azido-ATP cross-linking (compare lanes with DNA and those with adenosine in Fig. 1). Since dsDNA also stimulated the photo-cross-linking reaction, we further interpreted from these data that RecF protein binds dsDNA in the presence of ATP. Our data given below are consistent with this interpretation. The experiments described above were carried out at 0.5 ,uM RecF and 5.0 ,uM 8-azido-ATP. Under these conditions, 10 molecules of 8-azido-ATP were present for every molecule of RecF protein. If the interpretation given above, i.e., that binding of DNA increases RecF protein's ability to bind 8-azido-ATP, thereby resulting in the enhanced cross-linking signal, is correct, then we expect enhanced cross-linking in the presence of DNA even at other ratios of 8-azido-ATP to RecF protein. To test this, we carried out 8-azido-ATP cross-linking experiments in the presence of a fixed concentration of DNA but at different concentrations of 8-azidoATP. No adenosine control reaction was used in this experiment because no UV quenching at this concentration of DNA (i.e., 10 ,uM) was noted. For comparison, experiments were also done in the absence of DNA. Reaction samples after analysis on SDS-polyacrylamide gels were autoradiographed and scanned with a Hoefer densitometer. Peak intensities obtained at saturation were set at 100% for both DNA and no-DNA samples. The fractions of total saturation at different azido-ATP concentrations were then determined as shown in Fig. 2. Our results show that as the concentration of 8-azido-ATP increases in the absence of DNA, the fraction of total azido-ATP bound increases sigmoidally. In contrast, a linear increase followed by saturation of photo-cross-linking was noted in the presence of DNA (Fig. 2). This suggests that photo-cross-linking in the absence of DNA is cooperative, while cross-linking in the presence of DNA is not cooperative. Consistent with this interpretation, we noted that the
FIG. 2. Photo-cross-linking in the presence of different concentrations of 8-azido-ATP. RecF protein (0.5 p,M) was incubated with different amounts of 8-azido-ATP in the presence (*) and the absence (0) of ssDNA (5.0 F.M). All subsequent reaction conditions are as described in the legend to Fig. 1. Autoradiograms obtained were scanned with a Hoefer densitometer. From the peak values obtained, the fraction of total protein saturated was calculated.
absolute (i.e., not normalized) amount of photo-cross-linking at saturation is higher for reactions containing no DNA than for those containing DNA (data not shown). This suggests that in the absence of DNA, photo-cross-linking to both a DNA- and an ATP-binding site might be occurring, while in the presence of DNA, photo-cross-linking only to the ATPbinding site may occur. This suggestion could be tested by labeling and isolating the specific peptide(s) cross-linked with 8-azido-ATP for sequence analysis. From our data, it is difficult to determine the stoichiometry of ATP binding to RecF protein. We do not know what proportion of RecF monomers and/or 8-azido-ATP molecules is active in our experiments. Assuming that every RecF monomer and 8-azido-ATP molecule is active, then one can accurately determine the number of 8-azido-ATP molecules bound per RecF monomer (i.e., stoichiometry) at any given time both in the presence and in the absence of DNA. Our future experiments will address this problem. ATP competes with 8-azido-ATP cross-linking to RecF. An important criterion for determining whether 8-azido-ATP is photo-cross-linking at a specific site, e.g., an ATP-binding site, in RecF protein, is to ask whether natural analogs such as ATP, GTP, C1JP, and TJP can compete with 8-azido-ATP and thereby inhibit photo-cross-linking. If the photo-crosslinking is at a site which is nonspecific, then none of these natural analogs are expected to compete with 8-azido-ATP for cross-linking to RecF protein. To test this, we carried out photo-cross-linking experiments in the presence of different NTPs. Since the NTP used in the reactions might also inhibit photo-cross-linking by quenching UV light, parallel control reactions with adenosine were also carried out. Our results indicated that only ATP (Fig. 3) competed with 8-azido-ATP for binding to RecF. However, other NTPs such as GTP, CTP, UTP, and T at up to 500 mM did not compete with 8-azido-ATP for binding to RecF protein (data not shown). In Fig. 3, we can clearly see stimulation of photo-cross-
7708
MADIRAJU AND CLARK -ATP
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