Binding Of RecA Protein Research Articles

Quantitative genomic analysis of RecA protein binding during DNA double-strand break repair reveals RecBCD action in vivo

Understanding molecular mechanisms in the context of living cells requires the development of new methods of in vivo biochemical analysis to complement established in vitro biochemistry. A critically important molecular mechanism is genetic recombination, required for the beneficial reassortment of genetic information and for DNA double-strand break repair (DSBR). Central to recombination is the RecA (Rad51) protein that assembles into a spiral filament on DNA and mediates genetic exchange. Here we have developed a method that combines chromatin immunoprecipitation with next-generation sequencing (ChIP-Seq) and mathematical modeling to quantify RecA protein binding during the active repair of a single DSB in the chromosome of Escherichia coli. We have used quantitative genomic analysis to infer the key in vivo molecular parameters governing RecA loading by the helicase/nuclease RecBCD at recombination hot-spots, known as Chi. Our genomic analysis has also revealed that DSBR at the lacZ locus causes a second RecBCD-mediated DSBR event to occur in the terminus region of the chromosome, over 1 Mb away.

Effects of pressure and temperature on the binding of RecA protein to single-stranded DNA

The binding and polymerization of RecA protein to DNA is required for recombination, which is an essential function of life. We study the pressure and temperature dependence of RecA binding to single-stranded DNA in the presence of adenosine 5'-[γ-thio]triphosphate (ATP[γ-S]), in a temperature regulated high pressure cell using fluorescence anisotropy. Measurements were possible at temperatures between 5-60 °C and pressures up to 300 MPa. Experiments were performed on Escherichia coli RecA and RecA from a thermophilic bacteria, Thermus thermophilus. For E. coli RecA at a given temperature, binding is a monotonically decreasing and reversible function of pressure. At atmospheric pressure, E. coli RecA binding decreases monotonically up to 42 °C, where a sharp transition to the unbound state indicates irreversible heat inactivation. T. thermophilus showed no such transition within the temperature range of our apparatus. Furthermore, we find that binding occurs for a wider range of pressure and temperature for T. thermophilus compared to E. coli RecA, suggesting a correlation between thermophilicity and barophilicity. We use a two-state model of RecA binding/unbinding to extract the associated thermodynamic parameters. For E. coli, we find that the binding/unbinding phase boundary is hyperbolic. Our results of the binding of RecA from E. coli and T. thermophilus show adaptation to pressure and temperature at the single protein level.

Direct Evidence of the Role of ATPγS in the Binding of Single-Stranded Binding Protein (Escherichia coli) and RecA to Single-Stranded DNA

To gain insight into the influence of ATPγS on the competitive binding of RecA and single-stranded binding protein (SSB) on single-stranded DNA (ssDNA), AFM imaging was used to examine the three-dimensional structures of the different complexes formed by the binding of the two proteins on ssDNA in the presence and absence of ATPγS. In the presence of ATPγS, RecA attaches to ssDNA, displacing SSB, to form continuous binding regions that caused considerable elongation of the strand. When ATPγS is absent, RecA could not compete with SSB and only binds at a few sites that correspond to the vacancy in ssDNA left when SSB unbinds. These results provide direct evidence that, while SSB binding affinity to DNA is substantially higher than that of RecA, the presence of ATPγS is sufficient to alter the events and enable RecA coating of DNA.

SSB Protein Limits RecOR Binding onto Single-stranded DNA

The RecO and RecR proteins form a complex that promotes the nucleation of RecA protein filaments onto SSB protein-coated single-stranded DNA (ssDNA). However, even when RecO and RecR proteins are provided at optimal concentrations, the loading of RecA protein is surprisingly slow, typically proceeding with a lag of 10 min or more. The rate-limiting step in RecOR-promoted RecA nucleation is the binding of RecOR protein to ssDNA, which is inhibited by SSB protein despite the documented interaction between RecO and SSB. Full activity of RecOR is seen only when RecOR is preincubated with ssDNA prior to the addition of SSB. The slow binding of RecOR to SSB-coated ssDNA involves the C terminus of SSB. When an SSB variant that lacks the C-terminal 8 amino acids is used, the capacity of RecOR to facilitate RecA loading onto the ssDNA is largely abolished. The results are used in an expanded model for RecOR action.

Three-strand Exchange by the Escherichia coli RecA Protein Using ITP as a Nucleotide Cofactor: MECHANISTIC PARALLELS WITH THE ATP-DEPENDENT REACTION OF THE RecA PROTEIN FROM STREPTOCOCCUS PNEUMONIAE

The RecA protein from Escherichia coli promotes an ATP-dependent three-strand exchange reaction between a circular single-stranded DNA (ssDNA) and a homologous linear double-stranded (dsDNA). We have now found that under certain conditions, the RecA protein is also able to promote the three-strand exchange reaction using the structurally related nucleoside triphosphate, ITP, as the nucleotide cofactor. However, although both reactions are stimulated by single-stranded DNA-binding (SSB) protein, the ITP-dependent reaction differs from the ATP-dependent reaction in that it is observed only at low SSB protein concentrations, whereas the ATP-dependent reaction proceeds efficiently even at high SSB protein concentrations. Moreover, the circular ssDNA-dependent ITP hydrolysis activity of the RecA protein is strongly inhibited by SSB protein (suggesting that SSB protein displaces RecA protein from ssDNA when ITP is present), whereas the ATP hydrolysis activity is uninhibited even at high SSB protein concentrations (because RecA protein is resistant to displacement by SSB protein when ATP is present). These results suggest that SSB protein does not stimulate the ITP-dependent strand exchange reaction presynaptically (by facilitating the binding of RecA protein to the circular ssDNA substrate) but may act postsynaptically (by binding to the displaced strand that is generated when the circular ssDNA invades the linear dsDNA substrate). Interestingly, the mechanistic characteristics of the ITP-dependent strand exchange reaction of the E. coli RecA protein are similar to those of the ATP-dependent strand exchange reaction of the RecA protein from Streptococcus pneumoniae. These findings are discussed in terms of the relationship between the dynamic state of the RecA-ssDNA filament and the mechanism of the SSB protein-stimulated three-strand exchange reaction.

C-terminal Deletions of the Escherichia coli RecA Protein: CHARACTERIZATION OF IN VIVO AND IN VITROEFFECTS

A set of C-terminal deletion mutants of the RecA protein of Escherichia coli, progressively removing 6, 13, 17, and 25 amino acid residues, has been generated, expressed, and purified. In vivo, the deletion of 13 to 17 C-terminal residues results in increased sensitivity to mitomycin C. In vitro, the deletions enhance binding to duplex DNA as previously observed. We demonstrate that much of this enhancement involves the deletion of residues between positions 339 and 346. In addition, the C-terminal deletions cause a substantial upward shift in the pH-reaction profile of DNA strand exchange reactions. The C-terminal deletions of more than 13 amino acid residues result in strong inhibition of DNA strand exchange below pH 7, where the wild-type protein promotes a proficient reaction. However, at the same time, the deletion of 13-17 C-terminal residues eliminates the reduction in DNA strand exchange seen with the wild-type protein at pH values between 7.5 and 9. The results suggest the existence of extensive interactions, possibly involving multiple salt bridges, between the C terminus and other parts of the protein. These interactions affect the pK(a) of key groups involved in DNA strand exchange as well as the direct binding of RecA protein to duplex DNA.

Regulation of RecA Protein Binding to DNA by Opposing Effects of ATP and ADP on Inter-domain Contacts: Analysis by Urea-induced Unfolding of Wild-type and C-terminal Truncated RecA

RecA protein requires ATP and its hydrolysis to ADP to complete the DNA strand-exchange reaction. We investigated how the nucleotides activate RecA by examining their effect on urea-induced unfolding, which could reflect domain–domain contact of protein. RecA is folded into three continuous domains: the N-terminal, central and C-terminal domains. The fluorescence of tyrosine residues, which lie mainly in the central domain, was modified in 1–3 M urea, while the red shift of fluorescence peak of the tryptophan residues located in the C-terminal domain occurred only in 3–6 M urea. Thus, the C-terminal domain of RecA is unfolded after the central part unfolds. The change in intensity of tryptophan fluorescence without a large shift in the peak at low concentrations of urea suggests that there are weak interactions between the central and C-terminal domains. This is supported by our observation that RecA protein lacking the C-terminal tail unfolded at lower concentrations of urea than the entire RecA, and with clear transitions, unlike the entire RecA. ATP and its unhydrolyzable analog (ATPγS), which enhance the binding of RecA to DNA, facilitated the urea-induced change in RecA tryptophan fluorescence, while ADP, an antagonist of ATP, prevented the change. ATP probably weakens the domain–domain contact and facilitates the DNA binding, while ADP stabilizes the contact and inhibits it. Supporting this conclusion, the binding of RecA lacking the C-terminal tail to DNA was not inhibited by ADP, while that of the intact RecA was.

The RecOR proteins modulate RecA protein function at 5' ends of single-stranded DNA.

The Escherichia coli RecF, RecO and RecR pro teins have previously been implicated in bacterial recombinational DNA repair at DNA gaps. The RecOR-facilitated binding of RecA protein to single-stranded DNA (ssDNA) that is bound by single-stranded DNA-binding protein (SSB) is much faster if the ssDNA is linear, suggesting that a DNA end (rather than a gap) facilitates binding. In addition, the RecOR complex facilitates RecA protein-mediated D-loop formation at the 5' ends of linear ssDNAs. RecR protein remains associated with the RecA filament and its continued presence is required to prevent filament disassembly. RecF protein competes with RecO protein for RecR protein association and its addition destabilizes RecAOR filaments. An enhanced function of the RecO and RecR proteins can thus be seen in vitro at the 5' ends of linear ssDNA that is not as evident in DNA gaps. This function is countered by the RecF/RecO competition for association with the RecR protein.

Binding of SSB and RecA protein to DNA-containing stem loop structures: SSB ensures the polarity of RecA polymerization on single-stranded DNA.

Single-stranded DNA-binding proteins play an important role in homologous pairing and strand exchange promoted by the class of RecA-like proteins. It is presumed that SSB facilitates binding of RecA on to ssDNA by melting secondary structure, but direct physical evidence for the disruption of secondary structure by either SSB or RecA is still lacking. Using a series of oligonucleotides with increasing amounts of secondary structure, we show that stem loop structures impede contiguous binding of RecA and affect the rate of ATP hydrolysis. The electrophoretic mobility shift of a ternary complex of SSB-DNA-RecA and a binary complex of SSB-DNA are similar; however, the mechanism remains obscure. Binding of RecA on to stem loop is rapid in the presence of SSB or ATPgammaS and renders the complex resistant to cleavage by HaeIII, to higher amounts of competitor DNA or low temperature. The elongation of RecA filament in a 5' to 3' direction is halted at the proximal end of the stem. Consequently, RecA nucleates at the loop and cooperative binding propagates the RecA filament down the stem causing its disruption. The pattern of modification of thymine residues in the loop region indicates that RecA monomer is the minimum binding unit. Together, these results suggest that SSB plays a novel role in ensuring the directionality of RecA polymerization across stem loop in ssDNA. These observations have fundamental implications on the role of SSB in multiple aspects of cellular DNA metabolism.

Binding of double-stranded DNA by Escherichia coli RecA protein monitored by a fluorescent dye displacement assay.

We have developed a new assay to characterize the double-stranded DNA (dsDNA) binding properties of RecA protein. This assay is based on measurement of changes in the fluorescence of a 4',6-diamidino-2-phenylindole (DAPI)-dsDNA complex upon RecA protein binding. The binding of RecA protein to a complex of DAPI and dsDNA results in displacement of the bound DAPI, producing a decrease in the observed fluorescence. DAPI displacement is dependent on both RecA protein and ATP; dATP and, to a lesser extent, UTP and dCTP also support the DAPI displacement reaction, but dGTP, GTP, dITP and TTP do not. Binding stoichiometry for the RecA protein-dsDNA complex measured by DAPI displacement is 3 bp per RecA protein monomer in the presence of ATP. These results, taken together with data for mutant RecA proteins, suggest that this DAPI displacement assay monitors formation of the high affinity DNA binding state of RecA protein. Since this state of RecA protein defines the form of the nucleoprotein filament that is active in DNA strand exchange, these findings raise the possibility that the RecA protein-dsDNA filament may possess a homologous pairing capacity.

(GT) n Repetitive tracts affect several stages of RecA-promoted recombination

The effect of GT/CA dinucleotide repeat tracts on RecA-dependent homologous recombination was examined in vitro. By measuring the binding of RecA protein to oligonucleotides containing GT or CA repeats using the surface plasmon resonance (BIAcore), we show that the efficiency of RecA protein binding is sequence dependent: the protein binds to non-repetitive, poly(CA) or poly(GT) sequences with an increasing affinity. This preferential binding to repetitive sequences is specific for RecA protein and is not observed with the single-strand DNA binding (SSB) protein. Despite the fact that RecA filaments formed on GT tracts efficiently bind duplex DNAs, they are unable to promote stable joint formation. Moreover, strand exchange between a duplex DNA and a fully homologous single-stranded DNA (ssDNA) containing dinucleotide repeats, is inhibited as a function of the length of the repetitive tract. The number of molecules which underwent a complete strand exchange decreased from 100% to 80% and 30% for DNA containing seven, 16 and 39 GT repeats, respectively. The inhibition is less pronounced when the ssDNA contains CA instead of GT dinucleotide repeats. We propose that the high affinity of RecA protein for (CA) n or (GT) n tracts prevents strand exchange from progressing across such sequences. Thus, our results suggest that repetitive tracts of dinucleotides CA/GT could influence recombinational activity potentially leading to an increase in genomic rearrangements.

Binding of RecA to anti-parallel poly(dA) · 2poly(dT) triple helix DNA

Binding of RecA protein to conventional anti-parallel poly(dA) · 2poly(dT) triplex DNA has been studied using flow linear dichroism spectroscopy. The association requires the presence of cofactor analog adenosine 5′- O-3-thiotriphosphate (ATPγS) and occurs with a rate similar to that for the association of RecA to double-stranded poly(dA) · poly(dT) DNA. The binding of RecA to DNA stiffens the nucleotide chain, as evidenced from high orientation already at low shear rates, and the complex with triplex DNA appears to be at least as stiff as that with the duplex DNA. Therefore, the observation of a lower magnitude of the LD spectrum at 260 nm, in the triplex-RecA compared to the duplex-RecA complex, but retained magnitude of protein LD at 280 nm, indicates a markedly impaired orientation of nucleo-bases, possibly reflecting a perturbation by RecA on the third strand making its bases deviate strongly from perpendicularity. The circular dichroism spectrum, appearing immediately after dissociation of RecA by SDS, suggests an intact triplex structure, meaning that complexation with RecA has not dissociated the third strand. In conclusion, binding of RecA to triplex DNA does not modify the main organisation of the strands, but could affect the base-base interactions between them. Tilted bases could reflect a conformational change that RecA imposes also on the biological intermediate triplex structure to relax the base-base hydrogen bonding between the DNA strands.

Quantitative RecA protein binding to the hybrid duplex product of DNA strand exchange.

Following a DNA strand exchange reaction, RecA protein remains bound to the hybrid DNA product. DNA strand exchange reactions were carried out under optimal conditions in the presence of both RecA protein and SSB protein. As monitored by a sensitive DNA underwinding assay, all of the RecA protein present in the RecA nucleoprotein filament that initiates the strand exchange reaction can be accounted for on the hybrid DNA. As shown elsewhere, the SSB is bound to the displaced single DNA strand. Previous studies showed that RecA protein will dissociate from dsDNA when ADP levels build up, or transfer from dsDNA to ssDNA when the latter is not bound by SSB. The present work (done with ATP regeneration and SSB) shows that efficient strand exchange occurs in the absence of a net dissociation or transfer of RecA monomers from the filament. Such a dissociation or transfer is therefore not a mechanistic requirement for DNA strand exchange. The results provide evidence against some models proposed for the DNA strand exchange mechanism.

Characterization of RecA mediated homologous pairing on nitrocellulose membrane

Reactions between a single-stranded DNA (ssDNA) and a double-stranded DNA (dsDNA) provide an efficienty model to study RecA promoted homologous recombination. We have devised an assay in which the ssDNA is first bound to a nitrocellulose membrane. RecA protein is loaded on this membrane (loading step) which is then incubated with a labelled homologous dsDNA (incubation step). Since this assay can be used for study of mutant RecA proteins or RecA-like activities in crude extracts from other organisms, we have characterized the reaction promoted on the membrane. Under these new conditions, the reaction keeps the main characteristics observed with classical assays performed in solution: increasing NaCl concentration destabilized the RecA-DNA complex; ATPγS was required for formation of stable RecA-DNA complex, initiation of the reaction exhibits the same polarity as in classical assays, a complete strand exchange with a 44 bp long duplex oligonucleotide has been recorded under our conditions. Moreover, our results indicate that the binding of RecA protein itself to the nitrocellulose membrane did not impair its ability to promote homologous pairing. Pairing reactions involving long dsDNA (6407 bp) were more efficient with hydrolysable ATP than with ATPγS only when the ssDNA was bound to the membrane. Furthermore, ATP hydrolysis was not required when using short dsDNA (44 bp). These results constitute experimental support for a new role for the ATPase activity of RecA protein: the energy produced could favor the initiator of RecA mediated recombination involving long stretches of DNA which have restricted freedom to rotation.

Interaction of Escherichia coli RecA protein with ATP and its analogues.

Interactions of Escherichia coli RecA protein with ATP and its analogues in the absence of DNA were studied by circular dichroic (CD) spectroscopy. The binding of RecA protein to ATP increased the CD band of ATP at around 260 nm. The positive CD band of the RecA protein-ATP complex suggested that the bound ATP was in the anti conformation, in accord with X-ray crystallographic data [Story, R.M. and Steitz, T.A. (1992) Nature 355, 374-376]. At pH 7.5 and at 25 degrees C the dissociation constant (Kd) and thermodynamic parameters for the binding of ATP to RecA protein were 18 microM (delta G = -6.5 kcal.mol-1), delta H = 0 kcal.mol-1, and delta S = 22 cal.mol-1.K-1. A non-hydrolyzable ATP analogue, adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S), gave a spectral change similar to that of ATP. The Kd for this analogue, 22 microM, was very close to the Km of ATP. These results in the absence of single-stranded DNA were different from those obtained by kinetic analysis [Weinstock, G.M. et al. (1981) J. Biol. Chem. 256, 8850-8855], which indicated that the inhibition constant of ATP gamma S was much smaller than the Km of ATP in the presence of DNA. For other ATP analogues (dATP, ADP, and dADP), similar spectral changes were observed, and their Kd values ranged from 19 to 54 microM. UTP, dUTP, and TTP also gave CD spectral changes, but not AMP, GTP, dGTP, CTP, and dCTP.(ABSTRACT TRUNCATED AT 250 WORDS)

RecA binding to bulge- and mismatch-containing DNAs. Certain single base mismatches provide strong signals for RecA binding equal to multiple base bulges

Studies from several laboratories have demonstrated that RecA protein can recognize a variety of perturbations in the DNA helix. Here, using a nitrocellulose filter binding assay, it was observed that RecA bound to bulge-containing DNAs more effectively than non-bulged DNA. The degree of binding of RecA protein to bulged DNA was dependent on the conformation of the bulged bases and the kinking angles produced by the bulges as determined by the type and number of bases in the bulge. Although a single base mismatch does not kink DNA, RecA protein showed preferential binding to DNAs containing certain single base mismatches. An A.C mismatch flanked by A.T base pairs in a 28-base pair (bp) DNA facilitated the binding of RecA protein to the same high level as when the 28-bp DNA contained a 4-base cytosine bulge. Chemical probing techniques were used to examine the structure of DNA within the RecA filament. It was found that upon binding of RecA protein, the DNA helix becomes accessible over at least 14 bp, and the degree of sensitivity agrees with the binding efficiency of RecA protein.

Biochemical interaction of the Escherichia coli RecF, RecO, and RecR proteins with RecA protein and single-stranded DNA binding protein.

The Escherichia coli RecF, RecO, and RecR proteins were analyzed for their effect on RecA-mediated pairing of single-stranded circular DNA and homologous linear duplex DNA substrates. As shown by other workers, joint molecule formation by RecA was inhibited by E. coli single-stranded DNA binding protein (SSB) when it was added to single-stranded DNA before RecA. This inhibitory effect was overcome by the addition of RecO and RecR or RecF, RecO, and RecR. Both the rate and extent of joint molecule formation were restored to the maximal level observed when SSB was added after RecA. RecF, RecO, and RecR proteins had no effect on the conversion of joint molecules to final products and only appeared to stimulate an early step in the pairing reaction. The stimulatory effect of RecF, RecO, and RecR was not seen without SSB or when SSB was added after RecA. RecF protein by itself inhibited reactions in mixtures containing RecA and SSB, and this inhibition was overcome by the addition of RecO and RecR. These data suggest that RecO and RecR, and possibly RecF, help RecA overcome inhibition by SSB and utilize SSB-single-stranded-DNA complexes as substrates.

Use of structure-directed DNA ligands to probe the binding of recA protein to narrow and wide grooves of DNA and on its ability to promote homologous pairing.

We have used circular dichroism and structure-directed drugs to identify the role of structural features, wide and narrow grooves in particular, required for the cooperative polymerization, recognition of homologous sequences, and the formation of joint molecules promoted by recA protein. The path of cooperative polymerization of recA protein was deduced by its ability to cause quantitative displacement of distamycin from the narrow groove of duplex DNA. By contrast, methyl green bound to the wide groove was retained by the nucleoprotein filaments comprised of recA protein-DNA. Further, the mode of binding of these ligands and recA protein to DNA was confirmed by DNaseI digestion. More importantly, the formation of joint molecules was prevented by distamycin in the narrow groove while methyl green in the wide groove had no adverse effect. Intriguingly, distamycin interfered with the production of coaggregates between nucleoprotein filaments of recA protein-M13 ssDNA and naked linear M13 duplex DNA, but not with linear phi X174 duplex DNA. Thus, these data, in conjunction with molecular modeling, suggest that the narrow grooves of duplex DNA provide the fundamental framework required for the cooperative polymerization of recA protein and alignment of homologous sequences. These findings and their significance are discussed in relation to models of homologous pairing between two intertwined DNA molecules.

RecA protein-promoted homologous pairing and strand exchange between intact and partially single-stranded duplex DNA

In the pairing reaction between circular gapped and fully duplex DNA, RecA protein first polymerizes on the gapped DNA to form a nucleoprotein filament. Conditions that removed the formation of secondary structure in the gapped DNA, such as addition of Escherichia coli single-stranded DNA binding protein or preincubation in 1 m m-MgCl 2, optimized the binding of RecA protein and increased the formation of joint molecules. The gapped duplex formed stable joints with fully duplex DNA that had a 5′ or 3′ terminus complementary to the single-stranded region of the gapped molecule. However, the joints formed had distinct properties and structures depending on whether the complementary terminus was at the 5′ or 3′ end. Pairing between gapped DNA and fully duplex linear DNA with a 3′ complementary terminus resulted in strand displacement, symmetric strand exchange and formation of complete strand exchange products. By contrast, pairing between gapped and fully duplex DNA with a 5′ complementary terminus produced a joint that was restricted to the gapped region; there was no strand displacement or symmetric strand exchange. The joint formed in the latter reaction was likely a three-stranded intermediate rather than a heteroduplex with the classical Watson-Crick structure. We conclude that, as in the three-strand reaction, the process of strand exchange in the four-strand reaction is polar and progresses in a 5′ to 3′ direction with respect to the initiating strand. The present study provides further evidence that in both three-strand and four-strand systems the pairing and strand exchange reactions share a common mechanism.

The chemical mutagen dimethyl sulphate induces homologous recombination of plasmid DNA by increasing the binding of RecA protein to duplex DNA

The role of different DNA damages in the stimulation of homologous recombination was studied by using an in vivo plasmid recombination assay. Dimethyl sulphate (DMS) treatment of plasmid DNA induced a 20–50-fold increase in the frequency of recombinational events. DMS treatment also stimulated RecA protein binding to double-stranded DNA. In contrast, plasmid DNA containing uracil, which, like DMS, is also subject to repair, was less effective in stimulation of recombination. The ability of purified RecA protein to bind DMS-treated or uracil-containing DNA was tested by measuring its ATPase activity. The result indicates that DMS treatment, but not uracil incorportion, stimulates RecA protein binding to DNA. We conclude, that the main reason (or the first step) for stimulation of recombination by mutagens is activation of RecA binding to damaged DNA.