(Received for publication, August 22, 1995; and in revised form, October 11, 1995)
From the
To function as a repair and recombination protein, RecA has to
be assembled as an active filament on single-stranded DNA in the
presence of ATP or its analogs. We have identified amino acids in the
primary DNA binding site of RecA that interact with single-stranded DNA
by photocross-linking. A nucleoprotein complex consisting of RecA
protein bound to a monosubstituted oligonucleotide bearing a
5-iododeoxyuracil cross-linking moiety was irradiated with long
wavelength ultraviolet radiation to effect cross-linking with RecA
protein. Subsequent trypsin digestion, followed by purification and
peptide sequencing, revealed the cross-linking of two independent
peptides, amino acid residues 153-169 and 199-216.
Met from loop L1 and Phe
from loop L2 were
determined to be the exact points of cross-linking. Thus, our data
confirm and extend predictions about the DNA binding domain of RecA
protein based on the molecular structure of RecA (Story, R. M., Weber,
I. T., and Steitz, T. A.(1992) Nature 355, 318-325).
[Medline]
RecA protein is the central enzyme of bacterial homologous
recombination. It also plays an important role in DNA repair and the
SOS response (reviewed in (1, 2, 3, 4) ). The discovery of
eukaryotic RecA analogs Rad51 and DMC1, that have sequence homology to
RecA and, possibly, similar biochemical activities in
vitro(5, 6) , makes RecA protein a prototype of
this kind of enzyme in all organisms. RecA protein adopts an active
conformation only after its polymerization on ssDNA ()in the
presence of triphosphate nucleotide cofactor to form a presynaptic
filament. That this presynaptic filament is an essential precursor of
recombination reactions and the SOS response has led to a great
interest in the structure of these DNA
RecA complexes (reviewed in (7) ). This study addresses the position of ssDNA in the RecA
filament and the identification of amino acids that are in contact with
the ssDNA.
Photocross-linking has been used previously to determine
the exact amino acids contacting DNA in a number of DNA (or
RNA)-protein complexes (see (8, 9, 10) for
mechanism and references). Willis et al. (11) demonstrated that 5-iodouracil-substituted DNA or RNA can
be cross-linked with an extremely high yield by monochromatic 325-nm
irradiation. From analogy with 5-bromodeoxyuridine, we may expect that
UV irradiation of IdU with an emission peak at 312 nm can
result in high yields of photocross-linking with tryptophans,
tyrosines, and histidines and photocross-linking with lower yields with
most of the other amino acids.
In this report we show that two
loops, L1 and L2, of RecA protein are in close contact with
single-stranded DNA. We have determined Met of loop L1
and Phe
of loop L2 to be the exact positions of
photocross-linking.
RecA protein was isolated and purified as previously
described (12) . Oligonucleotides were synthesized by automated
-cyanoethyl phosphoramidite DNA synthesis on a 380B DNA
synthesizer (Applied Biosystems).
Irradiation in a UV Stratalinker 1800 (Stratagene), loaded with 312-nm bulbs (40 watts total) was carried out for 8 h. After irradiation the DNA-protein complex was dissociated by adding 15 mM EDTA, 5 mM ADP, 0.5 M NaCl, and 0.1% SDS.
A
small oligo(dT) cellulose column was poured. The column was
equilibrated with binding buffer (10 mM Tris-HCl, pH 8.0, 0.1
mM EDTA, 0.4 mM DTT, 0.1% SDS, 0.5 M NaCl)
prior to sample loading. Then it was washed with 10 volumes of binding
buffer, and the sample was eluted in elution buffer 1 (10 mM
Tris-HCl, pH 8.0, 0.05 mM EDTA, 0.4 mM DTT, 0.01%
SDS). DNA-containing fractions were identified by following the P label. SDS and DTT were adjusted to 0.3% and 3
mM, correspondingly, in a reaction volume of 0.5 ml. The tube
was heated at 85 °C for 20 min to denature the protein. After
denaturation, the reaction was allowed to cool, and 1 ml of clostripain
buffer (20 mM Tris-HCl, pH 7.5, 1 mM
CaCl
, 2 mM DTT) was added. After 4 h of
clostripain digestion with 40 µg of enzyme at 37 °C, an extra
40 µg of clostripain were added, and the cleavage was continued
overnight.
The sample was applied to oligo(dT) cellulose as
described above. The oligonucleotide was eluted with buffer 2 (10
mM Tris-HCl, pH 8.0, 0.05 mM EDTA, 0.4 mM DTT, 1 M urea) and concentrated six times in a Speedvac.
A small fraction was saved for a gel. The probe was heated at 85 °C
for 20 min. After denaturation the reaction was allowed to cool, and it
was diluted 6-fold with trypsin buffer (50 mM Tris-HCl, pH
7.5, 1 mM CaCl). After 4 h of modified trypsin
digestion with 40 µg of enzyme at 37 °C, an extra 40 µg of
trypsin were added, and the cleavage was continued overnight.
The sample was again purified by oligo(dT) cellulose chromatography and then purified by gel electrophoresis. The probe was loaded onto a 10% denaturing (urea) PAGE, and the bands were cut from the gel, eluted, and sent to the W. M. Keck Foundation Biotechnology Resource Laboratory at Yale University for peptide sequencing.
The
time course of the photocross-linking reaction showed that the reaction
reached saturation with 19% of oligonucleotides cross-linked after 4 h,
detected by gel-shift assay (Fig. 1, filled circles).
Most of the photocross-links occurred at the 5-iododeoxyuridine
position, as the cross-linking yield was more than 10 times less for a
control oligonucleotide containing deoxythymidine instead of IdU (Fig. 1, open circles).
Figure 1:
Time course of photocross-linking ssDNA
to RecA protein. 5`-end labeled oligonucleotides
d(A)CTATCTTAC (open circles) and
d(A)
CTATC(
IU)TAC (filled circles)
coated by RecA were UV irradiated. Aliquots at the indicated times were
loaded onto 10-20% Tricine-SDS minigels (NOVEX) followed by
PhosphorImager (Molecular Dynamics)
quantitation.
RecA protein
binds to DNA phosphates and it can form a presynaptic filament on any
sequence(16) . As the van der Waals radius of iodine was only
8% larger than the methyl group, we did not anticipate that the
monosubstitution of photoactive IdU for thymine would
significantly alter the structure of the presynaptic filament. Fig. 2provides additional evidence that the cross-linking signal
is specific. In the presence of ADP alone RecA protein cannot form a
stable complex with the
IdU-monosubstituted oligonucleotide
and UV irradiation did not result in the formation of any nonspecific
photocross-links (Fig. 2, lane 2). Furthermore, an
unsubstituted, unlabeled oligonucleotide of the same sequence competed
with the labeled
IdU-monosubstituted oligonucleotide for
the same binding sites on the RecA protein (Fig. 2, lanes
4-8). Thus, the incorporation of one
IdU does
not significantly alter the binding mode of the RecA protein. As
expected, the presynaptic filament formed on
IdU-monosubstituted DNA was able to find a homologous
sequence in a double-stranded plasmid DNA to form a synaptic complex
(data not shown). The incorporation of an adenine tail at the 5` end of
the oligonucleotide provided us with a simple and powerful strategy for
the purification of proteins and/or peptides covalently attached to the
oligo. Oligonucleotides bound to oligo(dT) cellulose at high ionic
strength (500 mM NaCl) and eluted in low salt conditions when
the oligo(dA-dT) duplex was destabilized. All uncross-linked
polypeptides passed through the column without trapping, which resulted
in very efficient purification of the sample.
Figure 2:
An excess of unmodified DNA competes out
the IdU-monosubstituted oligonucleotide from DNA binding
sites of RecA protein. The 5`-end-labeled oligonucleotide
d(A)
CTATC(
IU)TAC was incubated for 1 h with
RecA protein in the presence of different nucleotide cofactors and the
indicated excess of the unlabeled and unmodified oligonucleotide
d(A)
CTATCTTAC followed by UV irradiation for 4 h. Lane
1, oligonucleotide; lane 2, the reaction buffer contained
1.1 mM ADP; lane 3, the complex was formed in the
presence of 0.3 mM ATP
S and 1.1 mM ADP; lanes 4-8, 0.1 mM ATP
S was used as a
cofactor. Lanes 1-4, no competitive DNA was added; lanes 5-8, the molar excess of unsubstituted DNA added
to the reaction mixtures is indicated at the top.
We utilized a two-step endoprotease digestion to obtain a complete trypsin digestion and to minimize sample losses. The first step was cleavage of cross-linked protein in the presence of 0.1% SDS by clostripain, which cleaves at the carboxylic end of all arginines. The second step was redigestion of cross-linked peptides in the presence of 1 M urea by trypsin, which cleaves after both arginines and lysines. The products of these two rounds of protease digestion were resolved by electrophoresis on a 10% polyacrylamide urea gel. The oligonucleotide and cross-linked samples were detected by autoradiography.
We resolved three different cross-linked peptides (Fig. 3, lane 2). To check whether we obtained the shortest completely cleaved peptides we combined different endoproteinase digestions. A part of the sample after trypsin cleavage was saved for gel analysis (Fig. 3, lane 2), while the other part was repurified by oligo(dT) chromatography and then subjected to either clostripain (lane 3), trypsin (lane 4), or Staphylococcus aureus V8 protease (lanes 5-16) treatment. Clostripain did not change the cross-linked peptide pattern obtained (compare lanes 2 and 3). Additional trypsin treatment did not give rise to any new shorter products, but band C disappeared. Thus bands A and B contain the two shortest distinct peptides, while band C was an underdigested tryptic cross-linked peptide, that was converted into either A or B during the second round of trypsin cleavage. We expect the resulting peptides in samples A and B to be short because they migrated faster than samples cross-linked to a peptide 20 amino acids long (data not shown).
Figure 3: Superposition of endoprotease digestions. Lane 1, oligonucleotide; lane 2 cross-linked peptides from a two-step (clostripain/trypsin) digestion. Samples were redigested by clostripain (lane 3), trypsin (lane 4), or S. aureus V8 protease (lanes 5-16). Length of time of the third digestion is indicated at the top of the autoradiogram. The sizes of the cross-linked peptides determined by amino acid sequencing are in square brackets.
We also performed a time course of V8 protease cleavage of tryptic peptides in order to reveal the relationship between the three products (bands A, B, and C) and to determine whether these peptides have aspartic or glutamic acids in their sequence. The specificity of V8 protease depends on the buffer used for digestion. It cleaves specifically at the carboxylic side of glutamic and aspartic acid in 50 mM sodium phosphate, pH 7.8. In 50 mM ammonium bicarbonate, pH 7.8, this protease cleaves only after glutamic acid. Comparing the intensities of all the bands (lanes 5-16), it is clear that sample B was converted into B` and, presumably, has one glutamic acid. Band A was relatively resistant to V8 digestion, but had at least two sites of cleavage (Glu or Asp) leading to A`. Band C behaved as B, so it was reasonable to expect that sample C represents the underdigestion of band B (see Fig. 4for the peptide map).
Figure 4: Map of cross-linked peptides. Outlined R or K are potential sites of trypsin cleavage (arginine or lysine). Italicized E or D are potential sites of V8 protease cleavage (glutamic or aspartic acid). Shadowed M and F are the exact positions of photocross-linking (methionine 164 and phenylalanine 203).
Figure 5: Two-step endoprotease digestion prior to peptide sequencing. On the left, a cartoon of the strategy. On the right, an autoradiogram of the digestion steps. Lane 1, oligonucleotide; lane 2, cross-linked RecA protein-DNA complexes treated with clostripain (R); lane 3, cross-linked complexes after additional treatment with modified trypsin (R+K).
The digestion was less complete than for small quantities. So, in addition to the typical triple band pattern observed in the lane 2 of Fig. 3, there was an extra band whose mobility coincided with the band before the trypsin treatment (Fig. 5, lane 2). Presumably, this top band represented the partially digested product. However, materials from all four gel shifted bands as well as the band corresponding to free oligonucleotide were sequenced. There was no peptide material in the major band corresponding to unbound oligonucleotide. All other gel-shifted bands gave amino acid sequences.
The gel-shifted band A contains amino acid residues 153-169 (Fig. 4), corresponding to loop L1 with small flanking regions in the molecular structure of RecA(17) . The recovery of Met was only 5.8% that of the expected value; therefore, it is the probable site of cross-linking.
The peptide component of band B matched residues 199-217 of the RecA protein. It contains most of loop L2 and terminates at the nearest lysine on the carboxyl side. The yield of Phe in cycle 5 was only 21% of the anticipated level; thus, it appears to be a potential site of cross-linking. The remaining 20% of this peptide may be cross-linked at other positions along the peptide spanning residues 199-217, although the sensitivity of the sequencing was not sufficient to unambiguously determine these minor species. As we used long wavelength UV radiation resulting in minimum damage to biological substrates, it is unlikely that the decreased yield of phenylalanine results from photodestruction rather than photocross-linking. Also, as a control, we subjected a synthetic peptide derived from amino acids residues 193-212 to the same dose of UV radiation and did not observe any decrease of Phe level in the sequence (data not shown). Because the sequences of the cross-linked peptides A and B do not contain internal sites for cleavage by trypsin, they are the shortest possible tryptic peptides. This is consistent with the fact that the cross-linked peptides A and B represent limit digests.
Sequencing confirmed our preliminary
conclusions about the nature of band C. The product C had peptide
extending from residues 199-222 so C was indeed an underdigested
band B, and lysine 216 was the position of undercleavage (see Fig. 4). As in the case of B, Phe was determined
as the primary photocross-linking position.
The longest peptide, D, started upstream from loops L1 and L2 at residue 135. We sequenced only the first 22 amino acids from the amino end and detected no obvious position of cross-linking among these 22 amino acids. Thus, the simplest explanation is that lane D represents a partial digest of products A and B.
We can explain the electrophoretic mobilities of all peptide-DNA species in Fig. 3on the basis of their peptide lengths and electrical charges (see Fig. 4for peptide map). Sample A migrates faster than B, as it has one less amino acid and a -3 electrical charge of the peptide versus the uncharged peptide component of B. A presence of six extra amino acids slows down the mobility of C in comparison with B. Digestion with V8 protease cleaves product B into B`. B` has the shortest cross-linked peptide, which is nine amino acids long, and exhibits the fastest mobility. The lengths of the digestion products of A` based on the cleavage specificity of V8 protease and the known peptide sequence are predicted to be 15, 13, 11, and 9 amino acids long. As a result, the mobility of A` is somewhat faster than A, but slower than that of B`. The inability to obtain all four predicted cleavage products of A` and the relative resistance to the V8 cleavage of the other peptides could be the result of steric protection by the cross-linked oligonucleotide in the vicinity of these potential protease cleavage sites.
We have not
found any evidence for any additional points of cross-linking and all
peptides have been accounted for. Thus, we can conclude that: (i) there
are only two independent points of photocross-linking, (ii) peptides A
and B are the shortest products of tryptic digestion, and (iii)
cross-links occurred at Met and Phe
.
Our cross-linking data confirm a prediction deduced from crystallographic studies of RecA protein(17, 18) . Within the crystal, RecA protein is organized in a 83.7 Å pitch right-handed helix with six protomers per turn(17) . Assuming that this helix reflects the geometry of the DNA-RecA filament, and bearing in mind the fact that the DNA lies near the center(19) , Story et al. (17) proposed that loops L1 and L2 lining the cavity down the filament axis are involved in DNA binding. This prediction, based on the x-ray data obtained in the absence of DNA and triphosphate cofactors, is confirmed and extended by our finding that these loops constitute the ssDNA (primary) binding site in the active DNA-RecA filament.
The identity of amino
acids in the loop L2 is important for proper RecA function. In
vitro, the recA430 mutation (Gly
Ser) led to
a decrease in ssDNA binding affinity(20) . Two other mutations
(Glu
Gln and Gly
Ala)
exhibited a recA
phenotype in
vivo(21) . In vitro, these two mutants were
deficient in promoting both the self-cleavage of LexA repressor and the
DNA strand-exchange reaction(22) . The DNA strand-exchange
defects were correlated with an inability of mutant proteins to
displace SSB from DNA(22) . All of these data are consistent
with the participation of loop L2 in ssDNA binding.
Gardner et al.(23) unambiguously demonstrated that a peptide (amino acids 193-212) spanning loop L2, binds to ssDNA(23) . This observation combined with our cross-linking findings strongly argues for a direct participation of loop L2 in ssDNA binding.
While
this manuscript was in preparation a study was published (24) that also used photocross-linking to identify the ssDNA
binding domain of RecA. Surprisingly, the authors claimed two other
regions to be the DNA binding sites of RecA in the presence of
ATPS, the regions of residues 89-106 and 178-183. As
these peptides are located on the outside surface of the filament and
far from the central axis, these observations are not in accord with
the x-ray (17) and electron microscopy data(19) , which
place the DNA near that axis. Although the difference between our and
their data is puzzling, it could be a consequence of different
experimental designs. A lack of cross-links in loops L1 and L2 could be
the result of a less powerful cross-linking strategy. Oligo(dT) was
photocross-linked to RecA protein by short UV (254 nm)
irradiation(24) . Halogen-substituted uridines are more
reactive than thymines. Thus, it is conceivable that, even though the
oligo(dT) might have close contacts with loops L1 and L2, it might not
be cross-linkable in these regions. In addition, the two regions of
residues 89-106 and 178-183 could be part of the second DNA
binding site of RecA. As we used a significant excess of RecA protein
compared to the work of Morimatsu and Horii(24) , only the
primary DNA binding site was occupied, and this may explain why we did
not have any cross-links with these secondary regions.