(Received for publication, August 23, 1995; and in revised form, March 10, 1996)
From the
Photochemical cross-linking has been used to identify residues in the Escherichia coli RecA protein that are proximal to and may directly mediate binding of DNA. Ultraviolet irradiation promotes specific and efficient cross-linking of the RecA protein to poly(deoxythymidylic) acid. Cross-linked peptides remaining covalently attached to the polynucleotide following proteolytic digestion with trypsin correspond to amino acids 61-72, 178-183, and 233-243 of the RecA protein primary sequence. Their location and surface accessibility in the crystal structure, along with the behavior of various recA mutants, support the assignment of the cross-linked regions to the DNA binding site(s) of the RecA protein. Functional overlap of amino acids 61-72 with an element of the ATP binding site suggests a structural mechanism by which nucleotide cofactors allosterically affect the RecA nucleoprotein filament.
Genetic recombination is a universal occurrence that serves to generate genetic diversity, to preserve genomic integrity, and to ensure proper partitioning of chromosomes. Because of its basic role in DNA metabolism, elucidating the mechanism of this cellular process is of considerable importance. The product of the RecA gene is virtually indispensable to genetic recombination in Escherichia coli(1, 2) , and extensive analysis has revealed a unique and critical enzymatic activity: the RecA protein promotes the recognition and exchange of strands between homologous DNA molecules(3, 4, 5, 6) . Consequently, by providing a model system for investigating biochemical steps central to genetic recombination, characterization of the RecA protein has laid the foundation for the discovery of both structural (7, 8) and functional analogues not only from eubacteria but also from organisms ranging from bacteriophage to higher eukaryotes (for review see (9) and references therein).
Interaction of the RecA protein with DNA and a nucleotide triphosphate cofactor, such as ATP, is fundamental to its ability to catalyze homologous pairing and subsequent transfer of strands between a variety of DNA substrates in vitro(10, 11) . The functional species of the RecA protein is a helical nucleoprotein filament assembled through cooperative polymerization on either single-stranded DNA or duplex DNA containing a single-stranded gap or tail. Nucleoprotein filament formation requires stoichiometric amounts of the RecA protein and the binding of ATP to induce an active conformation in which the DNA assumes a highly extended and unwound state(12, 13, 14) . This unusual DNA conformation appears to be universal to the mechanism of genetic recombination because homologs such as the uvsX and Rad51 proteins from T4 bacteriophage and Saccharomyces cerevisiae, respectively, form complexes with DNA that are structurally (15, 16) and functionally analogous(17, 18) .
The manner by which the RecA nucleoprotein filament recognizes sequence homology within a duplex DNA target remains unknown. Understanding of this mechanism would be greatly facilitated by definition of those regions within the RecA protein polymer that interact with DNA and are consequently central to the recognition process. Despite the resolution of a crystal structure(19, 20) , the sites responsible for DNA binding within the RecA protein remain undefined. Structural and mutational analyses substantiate that the production of covalent linkages between nucleotide bases and amino acids through the action of ultraviolet irradiation is a general approach to probe the molecular interactions between species at the immediate interface of protein-nucleic acid complexes (for review see (21) and (22) and references therein). Covalent bond formation is thought to occur by a free radical mechanism in which a hydrogen atom from a favorably positioned amino acid is abstracted by a photoexcited nucleic acid base(21) . Although model studies demonstrate differences in photo reactivity(23) , it is presumed that all amino acids can in principle be cross-linked to nucleic acid bases through the absorption of ultraviolet light provided that the participating functional groups are in close proximity.
In this study, we employ photochemical cross-linking to discern those amino acid residues within the RecA nucleoprotein complex that are intimately associated with and may potentially mediate binding of single-stranded DNA. We show that specific cross-linking of the RecA protein to polydeoxythymidylic acid is efficient and saturates at an apparent stoichiometry consistent with direct single-stranded DNA binding studies. These studies both complement and extend the recently reported cross-linking of the RecA protein to oligonucleotides(24) . The peptides within the primary structure of the RecA protein that are covalently linked to poly(dT) have been identified, and their possible involvement in the binding of DNA is discussed. The cross-linking of a peptide that coincides with a portion of the nucleotide binding site may provide further insight as to how conformational changes in the RecA nucleoprotein filament elicited by the binding and hydrolysis of ATP are directly transduced to regions implicated in DNA binding.
RecA protein was purified from E. coli strain
JC12772 (25) using a modified preparative protocol ()based on spermidine precipitation(26) ; its
concentration was determined using an extinction coefficient of 2.7
10
M
cm
at 280 nm. Single-stranded polynucleotides, poly(dT) and poly(dA)
acids, were purchased from Pharmacia Biotech Inc. The concentration of
poly(dT) (average length, 300 nucleotides) and poly(dA) (average
length, 390 nucleotides) was determined using an extinction
coefficients of 8520 M
(nucleotide)
cm
at 260 nm and 8600 M
(nucleotide) cm
at 257 nm, respectively.
ATP
S (
)was purchased from Boehringer Mannheim and
dissolved as a concentrated stock at pH 7.5 in TE buffer (10 mM Tris-HCl, 1 mM EDTA); its concentration was determined
using an extinction coefficient of 1.54
10
M
cm
at 260 nm.
Figure 1: A, photochemical cross-linking of the RecA protein to poly(dT). Samples containing RecA protein and poly(dT) were prepared under conditions that either support (100 mM NaCl binding, lanes 1-6) or prohibit complex formation (1000 mM NaCl nonbinding, lanes 7-12) and were irradiated with shortwave UV light for the times indicated. B, proteolytic sensitivity of cross-linked RecA protein-poly(dT) complexes. After ultraviolet irradiation for 120 s, cross-linked complexes were incubated in the absence (lane 2) or the presence (lane 3) of trypsin. As a control, poly(dT) was also subjected to trypsin (lane 1).
It is conceivable that photoactivated species
that are generated in solution may collide to yield stable, yet
spurious, covalent complexes; however, cross-linking to poly(dT) in
either the absence or the presence of ATPS is not observed when
the RecA protein is denatured prior to irradiation, indicating that
only the native RecA protein can be cross-linked (data not shown). To
further address whether the observed cross-linking is due to a specific
interaction between the RecA protein and the single-stranded
polynucleotide, several control experiments were conducted. Similar to
most protein-nucleic acid complexes(29) , the stability of RecA
protein-ssDNA complexes decreases with increasing salt
concentrations(28) . In agreement, photoinduced cross-linking
of the RecA protein to poly(dT) is sensitive to the salt concentration
as ultraviolet irradiation under conditions that prohibit complex
formation (i.e. 1 M NaCl) fails to produce a species
with reduced electrophoretic mobility (Fig. 1A; lanes 7-12). Conversely, cross-linking to poly(dT) is
restored at 1 M NaCl in the presence of ATP
S (data not
shown) due to the induction of the high affinity single-stranded DNA
binding state of the RecA protein(28) . Finally, photoadducts
between the RecA protein and poly(dT) are not formed when either or
both of the macromolecules are irradiated separately before mixing
(data not shown).
Ultraviolet irradiation inhibits the single-stranded DNA-dependent ATP hydrolysis activity of the RecA protein in a dosage-dependent manner. As depicted in Fig. 2, equivalent amounts of ``background'' photoinactivation are observed when the RecA protein is irradiated either alone or in the presence of poly(dT) under conditions (1000 mM NaCl) that prohibit binding; however, inhibition is increased under conditions that permit formation and thus cross-linking of RecA protein-poly(dT) complexes. Therefore, photochemical cross-linking to polynucleotide results in a differential inhibition of the ATP hydrolysis activity of the RecA protein.
Figure 2: Effects of ultraviolet irradiation on the single-stranded DNA-dependent ATP hydrolysis activity of RecA protein. The ATP hydrolysis activity was measured for 30 µl-aliquots withdrawn from irradiation reactions that contained 10 µM RecA protein, either in the absence or the presence of 70 µM poly(dT), and either 100 mM or 1000 mM NaCl (see legend in figure). The percentage of ATP hydrolysis activity was determined by normalizing the observed rates of ATP hydrolysis measured for irradiated samples to those of nonirradiated samples.
As shown in Fig. 3A, irradiation
of RecA protein complexes formed with either poly(dT) or poly(dA)
causes a differential inhibition of its ATP hydrolysis activity
(defined as the inhibition caused by ultraviolet light under conditions
permitting complex formation (100 mM NaCl) minus that observed
under conditions prohibiting complex formation (1000 mM NaCl)). In each case, the amount of ultraviolet exposure required
to achieve maximal extents of differential inhibition (cross-linking)
is approximately 70 s and is consistent with that estimated for
poly(dT) using gel electrophoresis (Fig. 1A).
Furthermore, the greater extent of RecA protein cross-linking to
poly(dT) (16%) relative to poly(dA) (
4%) agrees with previous
studies that reveal thymine to be the most photoreactive nucleotide
base(21) . The quantitative relationship between photochemical
cross-linking of the RecA protein and inhibition of its ATP hydrolysis
activity is supported by chromatography studies that demonstrate that
20% of the total RecA protein elutes from a strong anion exchange
column associated with poly(dT) (data not shown). Therefore, these
results indicate that formation of photoadducts between RecA protein
and polynucleotides correlates to a loss in enzymatic activity. When
studied as a function of poly(dT) concentration (Fig. 3B), the differential inhibition of ATP
hydrolysis saturates at an apparent stoichiometry (7 ± 1
nucleotides per RecA protein monomer), which parallels that obtained
using an assay that directly measures the binding of the RecA protein
to single-stranded DNA(28, 30) . Because ATP
S
inhibits the ATP hydrolysis activity of the RecA protein and induces a
high affinity ssDNA binding state of the RecA protein stable out to
concentrations of NaCl greater than 2 M, neither ATP
hydrolysis nor ion exchange chromatography were used to characterize
cross-linking of the RecA protein to poly(dT) in the presence of the
relatively nonhydrolyzable cofactor.
Figure 3: Differential inhibition of ATP hydrolysis activity due to photochemically induced cross-linking between RecA protein and polynucleotides. ATP hydrolysis activity was measured, and differential inhibition was calculated as described under ``Materials and Methods.'' A, the type of polynucleotide influences the extent of RecA protein cross-linking. Samples containing 10 µM RecA protein and 70 µM nucleotides of either poly(dT) (triangles) or poly(dA) (squares) were exposed to ultraviolet irradiation for the times indicated. B, the extent of RecA protein cross-linking determined as a function of the poly(dT) concentration. Samples comprised of 10 µM RecA protein and various amounts of poly(dT) (0, 30, 50, 70, 90, and 150 µM nucleotides) were irradiated with ultraviolet light for 2 min.
Figure 4:
Regions
of RecA protein photochemically cross-linked to poly(dT). The
three-dimensional structure of the RecA protein monomer is represented
as a ribbon drawn along the peptide backbone(19) .
-Strands within the major central domain are numbered 1-8 according to their order of occurrence in the
primary sequence. Disordered loops between amino acids 157-164
(L1) and 195-209 (L2) are indicated by dashed lines. The
position of ADP (white) is defined by diffusion into the
crystal(20) . Modeling was done using RasMol Molecular
Visualization software (version 2.5). Tryptic peptides of the RecA
protein covalently cross-linked to poly(dT), either in the absence or
the presence of ATP
S, are labeled as follows: green,
Ile
-Val-Glu-Ile-Tyr-Gly-Pro-Glu-Ser-Ser-Gly-Lys
; red, Leu
-Ala-Gly-Asn-Leu-Lys
; blue,
Glu
-Gly-Glu-Asn-Val-Val-Gly-Ser-Glu-Thr-Arg
.
The amino acid residues in bold type above were identified through
amino acid sequencing.
Figure 5: Location of cross-linked regions within the RecA protein polymer. A space-filling representation of a single turn of the helical polymer formed by six RecA protein monomers in the crystal structure (19, 20) is shown as viewed parallel to (both directions, A and B) and perpendicular to (C) the helical axis (RasMol, version 2.5). Alternating monomers of the RecA protein are colored yellow and orange, whereas ADP is white. Cross-linked amino acids 61-72, lysine 183, and 233-243 of the RecA protein are highlighted in green, red, and blue, respectively.
In the
second peptide, a potential role for lysine 183 in the interaction with
DNA is implicated by our photochemical cross-linking data and that of
Morimatsu and Horii(24) . A previous sequence comparison among
a limited set of eubacterial RecA proteins implied that a positively
charged residue at position 183 may be essential to function because
substitutions for lysine are accompanied by the simultaneous change of
the next amino acid to arginine or lysine (see (7) for
compilation). Currently, a more extensive alignment of 62 RecA protein
sequences from a diverse range of bacterial sources indicates that
although lysine at residue 183 is not conserved, the most common amino
acid at position 184 is either lysine or arginine (39) ;
because exceptions to this conservation of amino acid 184 are most
common within enterobacteria, it is conceivable that both structural
and functional demands are preserved in the E. coli RecA
protein by the presence of a positive charge at the preceding amino
acid. Although such primary sequence relationships are suggestive, the
fact that single substitutions for residues at positions 183 or 184
have dramatic effects on RecA protein-promoted activities substantiates
the idea that this region is functionally significant. Substitution of
lysine 183 by methionine confers a recA phenotype and results in a protein that is diminished in both DNA
binding and DNA-dependent ATP hydrolysis, is sensitive to inhibition by
the E. coli single-stranded DNA binding protein, and is incapable of
promoting DNA strand exchange. (
)Moreover, mutation of the
adjacent residue, glutamine 184, to lysine in the RecA1202 protein
produces a variant that can be activated by either atypical nucleic
acid substrates, such as rRNA and tRNA, or alternate nucleotide
cofactors(40, 41) . Located on an exterior surface
that borders the helical groove of the RecA protein filament (Fig. 5C), amino acid residues 183 and 184 contribute
to the intermolecular association between adjacent polymers within the
crystal(19) . This observation lead to the proposal that
bundling of polymers may serve to regulate RecA protein binding to DNA (i.e. dissociation of bundles activates the RecA
protein)(19) . Although many aggregate forms of the RecA
protein have been documented(42, 43, 44) ,
the species fundamental to its interaction with DNA is unclear;
nevertheless, specific cross-linking of lysine 183 within the
interfilament contact region implies that binding to DNA is competitive
with the bundling of a DNA-free form of the RecA protein.
Whereas
initial residues(233-238) of the third identified region exhibit
little amino acid conservation, glycine 239 is invariant among
eubacterial RecA proteins (7, 8) and thus may allow
for favorable interactions between DNA phosphates and polypeptide
backbone amide NH groups. The terminal arginine residue (amino acid
243) is also conserved among eubacterial RecA
proteins(7, 8) , and its mutation to alanine results
in a RecA protein with a lower apparent affinity for single-stranded
DNA but that retains self-assembly properties indistinguishable from
the wild-type protein(45) . Furthermore, arginine 243 is
included in a region (residues 243-310) of the RecA protein
originally predicted to be important for interaction with DNA based on
a primary sequence alignment among several proteins that bind
cooperatively and with high affinity to single-stranded
DNA(46) . Based on structural analysis of filamentous
bacteriophage gene 5 proteins (47, 48) , a portion of
this region within the RecA protein (residues 243-257) is
proposed to be comprised of a set of antiparallel -sheets that
constitute a binding surface for single-stranded DNA, referred to as a
``DNA binding wing.'' Whereas arginine 243 is located within
one of the primarily parallel arranged
-sheets of the major
central domain of the RecA protein (Fig. 4), the balance of this
region (residues 247-257) is an interconnecting loop segment that
contributes to the extensive interface formed between monomers in the
RecA protein polymer(19) . Thus, although it may include a
portion of the DNA binding site of the RecA protein, any similarity of
this region (residues 243-257) to the DNA binding wing of other
single-stranded DNA binding proteins appears coincidental and is not
manifest in conservation of structure.