Initiation of genetic recombination and recombination-dependent replication.

Recombination initiates at double-stranded DNA breaks and at single-stranded DNA gaps. These DNA strand discontinuities can arise from DNA-damaging agents and from normal DNA replication when the DNA polymerase encounters an imperfection in the DNA template or another protein. The machinery of homologous recombination acts at these breaks and gaps to promote the events that result in gene recombination, as well as the reattachment of detached replication arms and the resumption of DNA replication. In Escherichia coli, these events require collaboration (RecA, RecBCD, RecFOR, RecQ, RuvABC and SSB proteins) and DNA replication (PriABC proteins and the DNA polymerases). The initial steps common to these recombination and recombination-dependent replication processes are reviewed.

Novel homologs of replication protein A in archaea: implications for the evolution of ssDNA-binding proteins.

In Bacteria and Eukarya, ssDNA-binding proteins are central to most aspects of DNA metabolism. Until recently, however, no counterpart of an ssDNA-binding protein had been identified in the third domain of life, Archaea. Here, we report the discovery of a novel type of ssDNA-binding protein in the genomes of several archaeons. These proteins, in contrast to all known members of this protein family, possess four conserved DNA-binding sites within a single polypeptide or, in one case, two polypeptides. This peculiar structural organization allows us to propose a model for the evolution of this class of proteins.

Direct Fluorescent Imaging of Translocation and Unwinding by Individual DNA Helicases.

The unique translocation and DNA unwinding properties of DNA helicases can be concealed by the stochastic behavior of enzyme molecules within the necessarily large populations used in ensemble experiments. With recent technological advances, the direct visualization of helicases acting on individual DNA molecules has contributed significantly to the current understanding of their mechanisms of action and biological functions. The combination of single-molecule techniques that enable both manipulation of individual protein or DNA molecules and visualization of their actions has made it possible to literally see novel and unique biochemical characteristics that were previously masked. Here, we describe the execution and use of single-molecule fluorescence imaging techniques, focusing on methods that include optical trapping in conjunction with epifluorescent imaging, and also surface immobilization in conjunction with total internal reflection fluorescence visualization. Combined with microchannel flow cells and microfluidic control, these methods allow individual fluorescently labeled protein and DNA molecules to be imaged and tracked, affording measurement of DNA unwinding and translocation at single-molecule resolution.

Negative co-dominant inhibition of recA protein function. Biochemical properties of the recA1, recA13 and recA56 proteins and the effect of recA56 protein on the activities of the wild-type recA protein function in vitro.

We have investigated the biochemical properties of several Escherichia coli mutant recA proteins that display a null phenotype. These are the recA1, recA13 and recA56 proteins, each of which carries a single missense mutation. These proteins all share a common defect which is the inability to adopt the high affinity DNA binding state normally elicited by the nucleotide cofactor ATP. Consequently, other than the ability to bind ssDNA, they possess none of the in vitro enzymatic activities of recA protein. However, each protein has characteristics that are unique, leading to the conclusion that the observed mutant phenotypes arise through fundamentally different mechanisms. Despite the magnitude of these defects, the recA56 protein is able to differentially inhibit various activities of wild-type recA protein. Incorporation of recA56 protein into a presynaptic filament with the wild-type recA protein does not affect the ability of the wild-type protein to hydrolyze ATP, as judged by the turnover number (kcat), provided that the ssDNA concentration is not limiting; however, the affinity of wild-type recA protein for ATP is lowered by the presence of recA56 protein. Similarly, the ability to cleave lexA protein is only modestly inhibited. However, both the ability to compete with SSB protein for ssDNA binding sites and the DNA strand exchange activity of wild-type recA protein are severely inhibited by the presence of recA56 protein. These results suggest that individual monomeric components of the recA protein-DNA filament are translated through protein-protein contacts to become macroscopic properties of the filament.

Biochemical characterization of a mutant recBCD enzyme, the recB2109CD enzyme, which lacks chi-specific, but not non-specific, nuclease activity.

RecBCD enzyme of Escherichia coli is a DNA helicase which also possesses ATP-dependent nuclease activities. We have purified a mutant recBCD enzyme, designated recB2109CD enzyme, and have examined the nuclease activities of this protein in vitro to determine whether any alteration in these activities is responsible for the recombination-deficient phenotype of the recB2109 strain. The recB2109CD enzyme possesses all of the non-specific nuclease activities (dsDNA exonuclease and ssDNA exo- and endonuclease) associated with wild-type recBCD enzyme although they are reduced approximately 2 to 3-fold relative to the wild-type enzyme. The ATP-dependent dsDNA exonuclease activity of recB2109CD enzyme requires significantly higher ATP concentrations for optimal activity when compared to the wild-type enzyme. The ATP-independent ssDNA endonuclease activity of the two enzymes is similar, but the ATP-stimulated ssDNA endonuclease and ATP-dependent ssDNA exonuclease activities of the mutant enzyme are reduced relative to those of wild-type recBCD enzyme. Despite its ability to degrade linear dsDNA non-specifically, recB2109CD enzyme lacks sequence-specific nicking activity at chi sites, which are hotspots for genetic recombination. Since this interaction with chi significantly attenuates the non-specific dsDNA exonuclease activity of wild-type recBCD enzyme, these results suggest that the non-specific dsDNA exonuclease activity of the mutant enzyme cannot be attenuated, with the consequence that a DNA substrate which is suitable for recombination is not produced.

The mutant recBCD enzyme, recB2109CD enzyme, has helicase activity but does not promote efficient joint molecule formation in vitro.

The Escherichia coli recB2109CD enzyme displays a defect in homologous recombination. In vitro, it possesses significant levels of non-specific nuclease activity but is deficient in chi-dependent nicking activity. To determine whether an alteration in helicase activity contributes further to its in vivo defect, the ability of recB2109CD enzyme to unwind dsDNA was examined. The mutant enzyme is able to unwind DNA but has a kcat which is one-third that of the wild-type enzyme. While the Km for DNA ends of the wild-type and mutant enzymes at low NaCl concentrations are essentially equivalent, the Km for ATP of recB2109CD enzyme is nearly six times greater. The processivity of unwinding (i.e. the average length of DNA unwound before recB2109CD enzyme dissociates from the DNA substrate) at 1 mM-Mg2+ ion and 1 mM-ATP is approximately 13 kb/end, whereas that of wild-type recBCD enzyme is 30 kb/end. In an assay which requires the co-ordinate actions of the recBCD, recA, and SSB proteins, joint molecule formation in the presence of recB2109CD enzyme is up to sixfold slower and proceeds to a lower extent than that mediated by the wild-type enzyme. We conclude that although the reduced helicase activity of the mutant recBCD enzyme may contribute to its recombination deficiency, its defect in the chi-dependent attenuation of non-specific nuclease activity is primarily responsible for the recombination-deficiency of E. coli strains bearing the recB2109 mutation.

The simultaneous binding of two double-stranded DNA molecules by Escherichia coli RecA protein.

We have characterized the double-stranded DNA (dsDNA) binding properties of RecA protein, using an assay based on changes in the fluorescence of 4',6-diamidino-2-phenylindole (DAPI)-dsDNA complexes. Here we use fluorescence, nitrocellulose filter-binding, and DNase I-sensitivity assays to demonstrate the binding of two duplex DNA molecules by the RecA protein filament. We previously established that the binding stoichiometry for the RecA protein-dsDNA complex is three base-pairs per RecA protein monomer, in the presence of ATP. In the presence of ATPgammaS, however, the binding stoichiometry depends on the MgCl2 concentration. The stoichiometry is 3 bp per monomer at low MgCl2 concentrations, but changes to 6 bp per monomer at higher MgCl2 concentrations, with the transition occurring at approximately 5 mM MgCl2. Above this MgCl2 concentration, the dsDNA within the RecA nucleoprotein complex becomes uncharacteristically sensitive to DNase I digestion. For these reasons we suggest that, at the elevated MgCl2 conditions, the RecA-dsDNA nucleoprotein filament can bind a second equivalent of dsDNA. These results demonstrate that RecA protein has the capacity to bind two dsDNA molecules, and they suggest that RecA or RecA-like proteins may effect homologous recognition between intact DNA duplexes.

Interaction of recA protein with single-stranded DNA. Quantitative aspects of binding affinity modulation by nucleotide cofactors.

We have investigated quantitative molecular aspects of the interaction of recA protein with single-stranded DNA, by using a fluorescent modified-DNA referred to as etheno-M13 DNA. In addition, the effects of the nucleotide cofactors ATP and ADP, and the analogues ATP-gamma-S, AMP-P-C-P, and AMP-P-N-P on this interaction have been studied. It is shown that ATP, AMP-P-N-P and, in particular, ATP-gamma-S significantly increase the affinity of recA protein for single-stranded DNA, whereas ADP and, to a lesser degree, AMP-P-C-P decrease the affinity. Binding to etheno-M13 single-stranded DNA is co-operative, with the value of the co-operativity parameter, omega, being approximately 50 under all conditions measured. The effect that ADP has on recA protein-DNA affinity is to lower the intrinsic binding constant, but it has no effect on the co-operativity of binding. In addition, the stability of the recA protein-DNA complex is very salt dependent (d log K/d log [NaC1] approximately -10) and it is the intrinsic binding affinity rather than the co-operativity of binding that is affected; thus, under all conditions observed, recA protein binds single-stranded DNA co-operatively with a value of omega = 50 +/- 10. The binding affinity is also influenced by the type of anion present, being approximately 10,000-fold higher when acetate ion is present instead of chloride ion. These data have been interpreted to suggest that recA protein forms up to five ionic interactions when it binds to single-stranded DNA and that five to six anions are displaced upon binding. The modulation of recA protein-DNA complex stability by nucleotide cofactors suggests that these cofactors play a role in the cycling of recA protein on and off single-stranded DNA, with ATP being required for DNA binding under physiological conditions and ADP serving as a "release" factor. These results are discussed in terms of a model for the role of ATP hydrolysis in a recA protein-single stranded DNA binding cycle.

Biochemical basis of the temperature-inducible constitutive protease activity of the RecA441 protein of Escherichia coli.

We compared the biochemical properties of the RecA441 protein to those of the wild-type RecA protein in an effort to account for the constitutive protease activity observed in recA441 strains. The two RecA proteins have similar properties in the absence of single-stranded DNA binding protein (SSB protein), and the differences that do exist shed little light on the temperature-inducible phenotype observed in recA441 strains. In contrast, several biochemical differences are apparent when the two proteins are compared in the presence of SSB protein, and these are conducive to a hypothesis that explains the temperature-sensitive behavior observed in these strains. We find that both the single-stranded DNA (ssDNA)-dependent ATPase and LexA-protease activities of RecA441 protein are more resistant to inhibition by SSB protein than are the activities of the wild-type protein. Additionally, the RecA441 protein is more capable of using ssDNA that has been precoated with SSB protein as a substrate for ATPase and protease activities, implying that RecA441 protein is more proficient at displacing SSB protein from ssDNA. The enhanced SSB protein displacement ability of the RecA441 protein is dependent on elevated temperature. These observations are consistent with the hypothesis that the RecA441 protein competes more efficiently with SSB protein for limited ssDNA sites and can be activated to cleave repressors at elevated temperature by displacing SSB protein from the limited ssDNA that occurs naturally in Escherichia coli. Neither the ssDNA binding characteristics of the RecA441 protein nor the rate at which it transfers from one DNA molecule to another provides an explanation for its enhanced activities, leading us to conclude that kinetics of RecA441 protein association with DNA may be responsible for the properties of the RecA441 protein.

Effects of Escherichia coli SSB protein on the single-stranded DNA-dependent ATPase activity of Escherichia coli RecA protein. Evidence that SSB protein facilitates the binding of RecA protein to regions of secondary structure within single-stranded DNA.

The effect that Escherichia coli single-stranded DNA binding (SSB) protein has on the single-stranded DNA-dependent ATPase activity of RecA protein is shown to depend upon a number of variables such as order of addition, magnesium concentration, temperature and the type of single-stranded DNA substrate used. When SSB protein is added to the DNA solution prior to the addition of RecA protein, a significant inhibition of ATPase activity is observed. Also, when SSB protein is added after the formation of a RecA protein-single-stranded DNA complex using either etheno M13 DNA, poly(dA) or poly(dT), or using single-stranded phage M13 DNA at lower temperature (25 degrees C) and magnesium chloride concentrations of 1 mM or 4 mM, a time-dependent inhibition of activity is observed. These results are consistent with the conclusion that SSB protein displaces the RecA protein from these DNA substrates, as described in the accompanying paper. However, if SSB protein is added last to complexes of RecA protein and single-stranded M13 DNA at elevated temperature (37 degrees C) and magnesium chloride concentrations of 4 mM or 10 mM, or to poly(dA) and poly(dT) that was renatured in the presence of RecA protein, no inhibition of ATPase activity is observed; in fact, a marked stimulation is observed for single-stranded M13 DNA. A similar effect is observed if the bacteriophage T4-coded gene 32 protein is substituted for SSB protein. The apparent stoichiometry of DNA (nucleotides) to RecA protein at the optimal ATPase activity for etheno M13 DNA, poly(dA) and poly(dT) is 6(+/- 1) nucleotides per RecA protein monomer at 4 mM-MgCl2 and 37 degrees C. Under the same conditions, the apparent stoichiometry obtained using single-stranded M13 DNA is 12 nucleotides per RecA protein monomer; however, the stoichiometry changes to 4.5 nucleotides per RecA protein monomer when SSB protein is added last. In addition, a stoichiometry of four nucleotides per RecA protein can be obtained with single-stranded M13 DNA in the absence of SSB protein if the reactions are carried out in 1 mM-MgCl2. These data are consistent with the interpretation that secondary structure within the natural DNA substrate limits the accessibility of RecA protein to these regions. The role of SSB protein is to eliminate this secondary structure and allow RecA protein to bind to these previously inaccessible regions of the DNA.(ABSTRACT TRUNCATED AT 400 WORDS)

Biochemical events essential to the recombination activity of Escherichia coli RecA protein. II. Co-dominant effects of RecA142 protein on wild-type RecA protein function.

In the accompanying paper, RecA142 protein was found to be completely defective in DNA heteroduplex formation. Here, we show that RecA142 protein not only is defective in this activity but also is inhibitory for certain activities of wild-type RecA protein. Under appropriate conditions, RecA142 protein substantially inhibits the DNA strand exchange reaction catalyzed by wild-type RecA protein; at equimolar concentrations of each protein, formation of full-length gapped duplex DNA product molecules is less than 7% of the amount produced by wild-type protein alone. Inhibition by RecA142 protein is also evident in S1 nuclease assays of DNA heteroduplex formation, although the extent of inhibition is less than is observed for the complete DNA strand exchange process; at equimolar concentrations of wild-type and mutant proteins, the extent of DNA heteroduplex formation is 36% of the wild-type protein level. This difference implies that RecA142 protein prevents, at minimum, the branch migration normally observed during DNA strand exchange. RecA142 protein does not inhibit either the single-strand (ss) DNA-dependent ATPase activity or the coaggregation activities of wild-type RecA protein. This suggests that these reactions are not responsible for the inhibition of wild-type protein DNA strand exchange activity by RecA142 protein. However, under conditions where RecA142 protein inhibits DNA strand exchange activity, RecA142 protein renders the M13 ssDNA-dependent ATPase activity of wild-type protein sensitive to inhibition by single-strand DNA-binding protein, and it inhibits the double-strand DNA-dependent ATPase activity of wild-type RecA protein. These results imply that these two activities are important components of the overall DNA strand exchange process. These experiments also demonstrate the applicability of using defective mutant RecA proteins as specific codominant inhibitors of wild-type protein activities in vitro and should be of general utility for mechanistic analysis of RecA protein function both in vitro and in vivo.

Identification of the RecA protein-loading domain of RecBCD enzyme.

Genetic recombination in Escherichia coli is stimulated by the recombination hotspot Chi (chi), a regulatory element that modifies the activities of the RecBCD enzyme and leads to loading of the DNA strand exchange protein, RecA, onto the chi-containing DNA strand. The RecBC enzyme, which lacks the RecD subunit, loads RecA protein constitutively, in a manner that is independent of chi. Using a truncated RecBC enzyme lacking the 30 kDa C-terminal domain of the RecB subunit, we show that this domain is necessary for RecA protein-loading. We propose that this domain harbors a site that interacts with RecA protein, recruiting it to single-stranded DNA during unwinding. This ability of a translocating enzyme to deliver material (RecA protein) to a specific target site (the chi sequence) parallels that of other cellular motor proteins.

SSB protein controls RecBCD enzyme nuclease activity during unwinding: a new role for looped intermediates.

The RecBCD enzyme of Escherichia coli initiates homologous recombination by unwinding and simultaneously degrading DNA from a double-stranded DNA end. Single-stranded DNA loops are intermediates of this unwinding process. Here we show that SSB protein reduces the level of DNA degradation by RecBCD enzyme during unwinding, by binding to these ssDNA intermediates. Prior to interaction with the recombination hot spot chi, RecBCD enzyme has both 3'-->5' exonuclease and a weaker 5'-->3' exonuclease activity. We show that degradation of the 5'-terminal strand at the entry site is much more extensive in the absence of SSB protein. After interaction with chi, the level of 5'-->3' exonuclease activity is increased; as expected, degradation of the 5'-strand is also elevated in the absence of SSB protein. Furthermore, we show that, in the absence of SSB protein, the RecBCD enzyme is inhibited by the ssDNA products of unwinding; SSB protein alleviates this inhibition. These results provide insight into the organization of helicase and nuclease domains within the RecBCD enzyme, and also suggest a new level at which the nuclease activity of RecBCD enzyme is controlled. Hence, they offer new insight into the role of SSB protein in the initiation phase of recombination.

Biochemical properties of the Escherichia coli recA430 protein. Analysis of a mutation that affects the interaction of the ATP-recA protein complex with single-stranded DNA.

The biochemical properties of the recA430 protein have been examined and compared to those of wild-type recA protein. We find that, while the recA430 protein possesses ssDNA-dependent rATP activity, this activity is inhibited by the Escherichia coli single-stranded DNA binding protein (SSB protein) under many conditions that enhance wild-type recA protein rATPase hydrolysis. Stimulation of rATPase activity by SSB protein is observed only at high concentrations of both rATP (greater than 1 mM) and recA430 protein (greater than 5 microM). In contrast, stimulation of ssDNA-dependent dATPase activity by SSB protein is less sensitive to protein and nucleotide concentration. Consistent with the nucleotide hydrolysis data, recA430 protein can carry out DNA strand exchange in the presence of either rATP or dATP. However, in the presence of rATP, both the rate and the extent of DNA strand exchange by recA430 protein are greatly reduced compared to wild-type recA protein and are sensitive to recA430 protein concentration. This reduction is presumably due to the inability of recA430 protein to compete with SSB protein for ssDNA binding sites under these conditions. The cleavage of lexA repressor protein by recA430 protein is also sensitive to the nucleotide cofactor present and is completely inhibited by SSB protein when rATP is the cofactor but not when dATP is used. Finally, the steady-state affinity and the rate of association of the recA430 protein-ssDNA complex are reduced, suggesting that the mutation affects the interaction of the ATP-bound form of recA protein with ssDNA. This alteration is the likely molecular defect responsible for inhibition of recA430 protein rATP-dependent function by SSB protein. The biochemical properties observed in the presence of dATP and SSB protein, i.e. the reduced levels of both DNA strand exchange activity and cleavage of lexA repressor protein, are consistent with the phenotypic behavior of recA430 mutations.

The Bacillus subtilis AddAB helicase/nuclease is regulated by its cognate Chi sequence in vitro.

The AddAB enzyme is important to homologous DNA recombination in Bacillus subtilis, where it is thought to be the functional counterpart of the RecBCD enzyme of Escherichia coli. In vivo, AddAB responds to a specific five-nucleotide sequence (5'-AGCGG-3' or its complement) in a manner analogous to the response of the RecBCD enzyme to interaction with chi sequences. Here, we show that purified AddAB enzyme is able to load at a double-stranded DNA end and is both a DNA helicase and nuclease, whose combined action results in the degradation of both strands of the DNA duplex. During translocation, recognition of the properly oriented sequence 5'-AGCGG-3' causes attenuation of the AddAB enzyme nuclease activity that is responsible for degradation of the strand 3'-terminal at the entry site. Therefore, we conclude that 5'-AGCGG-3' is the B. subtilis Chi site and it is hereafter referred to as chi(Bs). After encountering chi(Bs), both the degradation of the 5'-terminal strand and the helicase activity persist. Thus, processing of a double-stranded DNA end by the AddAB enzyme produces a duplex DNA molecule with a protruding 3'-terminated single-stranded tail, a universal intermediate of the recombination process.

Effects of the Escherichia coli SSB protein on the binding of Escherichia coli RecA protein to single-stranded DNA. Demonstration of competitive binding and the lack of a specific protein-protein interaction.

The effect of the Escherichia coli single-stranded DNA binding (SSB) protein on the stability of complexes of E. coli RecA protein with single-stranded DNA has been investigated through direct DNA binding experiments. The effect of each protein on the binding of the other to single-stranded DNA, and the effect of SSB protein on the transfer rate of RecA protein from one single-stranded DNA molecule to another, were studied. The binding of SSB protein and RecA protein to single-stranded phage M13 DNA is found to be competitive and, therefore, mutually exclusive. In the absence of a nucleotide cofactor, SSB protein binds more tightly to single-stranded DNA than does RecA protein, whereas in the presence of ATP-gamma-S, RecA protein binds more tightly than SSB protein. In the presence of ATP, an intermediate result is obtained that depends on the type of DNA used, the temperature, and the magnesium ion concentration. When complexes of RecA protein, SSB protein and single-stranded M13 DNA are formed under conditions of slight molar excess of single-stranded DNA, no effect of RecA protein on the equilibrium stability of the SSB protein-single-stranded DNA complex is observed. Under similar conditions, SSB protein has no observed effect on the stability of the RecA protein-etheno M13 DNA complex. Finally, measurements of the rate of RecA protein transfer from RecA protein-single-stranded DNA complexes to competing single-stranded DNA show that there is no kinetic stabilization of the RecA protein-etheno M13 DNA complex by SSB protein, but that a tenfold stabilization is observed when single-stranded M13 DNA is used to form the complex. However, this apparent stabilizing effect of SSB protein can be mimicked by pre-incubation of the RecA protein-single-stranded M13 DNA complex in low magnesium ion concentration, suggesting that this effect of SSB protein is indirect and is mediated through changes in the secondary structure of the DNA. Since no direct effect of SSB protein is observed on either the equilibrium or dissociation properties of the RecA protein-single-stranded DNA complex, it is concluded that the likely effect of SSB protein in the strand assimilation reaction is on a slow step in the association of RecA protein with single-stranded DNA. Direct evidence for this conclusion is presented in the accompanying paper.

Biochemical events essential to the recombination activity of Escherichia coli RecA protein. I. Properties of the mutant RecA142 protein.

We have characterized the biochemical properties of Escherichia coli RecA142 protein, the product of a recA allele that is phenotypically defective in genetic recombination. In vitro, this mutant RecA protein is totally defective in DNA heteroduplex formation. Despite this defect, RecA142 protein is not deficient in all other biochemical activities. RecA142 protein is proficient in single-strand (ss) DNA binding ability, ssDNA-dependent ATPase activity, and DNA-free self-association (although the first 2 properties show a greater sensitivity to NaCl concentration than does the wild-type protein). However, RecA142 protein is deficient in four properties: (1) its ssDNA-dependent ATPase activity is completely inhibited by ssDNA binding (SSB) protein, demonstrating that RecA142 protein is unable to compete effectively with SSB protein for ssDNA binding sites; (2) it is unable to promote the coaggregation of ssDNA and double-strand (ds) DNA; (3) its M13 dsDNA-dependent ATPase activity is attenuated to approximately 5% of the level of the wild-type protein; (4) it is unable fully to develop characteristics of the high-affinity ssDNA-binding state that is normally induced by ATP. The first three deficiencies correspond to defects in the presynaptic, synaptic and postsynaptic steps of the in vitro DNA strand exchange reaction, respectively; the fourth is the likely fundamental basis for defects 1 and 3. Therefore, one or more of these properties must be important to both the in vitro and in vivo processes.

A novel, 11 nucleotide variant of chi, chi*: one of a class of sequences defining the Escherichia coli recombination hotspot chi.

In wild-type Escherichia coli, recognition of the recombination hotspot, chi (5'-GCTGGTGG-3'), by the RecBCD enzyme is central to homologous recombination. However, in the recC* class of RecBCD mutants, stimulation of recombination by the canonical chi sequence is not detectable, but the levels of homologous recombination are nearly wild-type. In vivo studies demonstrate that a member of this class of mutants, the recC1004 allele, encodes an enzyme that responds to a novel variant of chi, termed chi* (5'-GCTGGTGCTCG-3'). Here, we establish that, in vitro, the chi* sequence is recognized more efficiently by the RecBC(1004)D enzyme than is the wild-type chi. This is manifest by both a greater modification of nuclease activity and a higher stimulation of RecA protein-mediated joint molecule formation at chi* than at chi. Sequencing of the recC1004 gene revealed that it contains a frameshift mutation, which results in a replacement of nine of the wild-type amino acid residues by eight in the mutant protein, and defines a locus that is important for the specificity of chi-recognition. In addition, we show that this novel, 11 nucleotide chi* sequence also regulates the wild-type RecBCD enzyme, supporting the notion that variants of the canonical chi constitute a class of sequences that regulate the recombination function of RecBCD enzyme.

Archaeal RadA protein binds DNA as both helical filaments and octameric rings.

The Escherichia coli RecA protein has been a model for understanding homologous eukaryotic recombination proteins such as Rad51. The active form of both RecA and Rad51 appear to be helical filaments polymerized on DNA, in which an unusual helical structure is induced in the DNA. Surprisingly, the human meiosis-specific homolog of RecA, Dmc1, has thus far only been observed to bind DNA as an octameric ring. Sequence analysis and biochemical studies have shown that archaeal RadA proteins are more closely related to Rad51 and Dmc1 than the bacterial RecA proteins. We find that the Sulfolobus solfataricus RadA protein binds DNA in the absence of nucleotide cofactor as an octameric ring and in the presence of ATP as a helical filament. Since it is likely that RadA is closely related to a common ancestral protein of both Rad51 and Dmc1, the two DNA-binding forms of RadA may provide insight into the divergence that has taken place between Rad51 and Dmc1.

Rad54 protein stimulates heteroduplex DNA formation in the synaptic phase of DNA strand exchange via specific interactions with the presynaptic Rad51 nucleoprotein filament.

RAD54 is an important member of the RAD52 group of genes that carry out recombinational repair of DNA damage in the yeast Saccharomyces cerevisiae. Rad54 protein is a member of the Snf2/Swi2 protein family of DNA-dependent/stimulated ATPases, and its ATPase activity is crucial for Rad54 protein function. Rad54 protein and Rad54-K341R, a mutant protein defective in the Walker A box ATP-binding fold, were fused to glutathione-S-transferase (GST) and purified to near homogeneity. In vivo, GST-Rad54 protein carried out the functions required for methyl methanesulfonate sulfate (MMS), UV, and DSB repair. In vitro, GST-Rad54 protein exhibited dsDNA-specific ATPase activity. Rad54 protein stimulated Rad51/Rpa-mediated DNA strand exchange by specifically increasing the kinetics of joint molecule formation. This stimulation was accompanied by a concurrent increase in the formation of heteroduplex DNA. Our results suggest that Rad54 protein interacts specifically with established Rad51 nucleoprotein filaments before homology search on the duplex DNA and heteroduplex DNA formation. Rad54 protein did not stimulate DNA strand exchange by increasing presynaptic complex formation. We conclude that Rad54 protein acts during the synaptic phase of DNA strand exchange and after the formation of presynaptic Rad51 protein-ssDNA filaments.