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.

RecA protein filaments disassemble in the 5' to 3' direction on single-stranded DNA.

RecA protein forms filaments on both single- and double-stranded DNA. Several studies confirm that filament extension occurs in the 5' to 3' direction on single-stranded DNA. These filaments also disassemble in an end-dependent fashion, and several indirect observations suggest that the disassembly occurs on the end opposite to that at which assembly occurs. By labeling the 5' end of single-stranded DNA with a segment of duplex DNA, we demonstrate unambiguously that RecA filaments disassemble uniquely in the 5' to 3' direction.

RecA protein promotes the regression of stalled replication forks in vitro.

Replication forks are halted by many types of DNA damage. At the site of a leading-strand DNA lesion, forks may stall and leave the lesion in a single-strand gap. Fork regression is the first step in several proposed pathways that permit repair without generating a double-strand break. Using model DNA substrates designed to mimic one of the known structures of a fork stalled at a leading-strand lesion, we show here that RecA protein of Escherichia coli will promote a fork regression reaction in vitro. The regression process exhibits an absolute requirement for ATP hydrolysis and is enhanced when dATP replaces ATP. The reaction is not affected by the inclusion of the RecO and R proteins. We present this reaction as one of several potential RecA protein roles in the repair of stalled and/or collapsed replication forks in bacteria.

New insights into protein-DNA interactions obtained by electron microscopy.

The electron microscopic study of DNA-protein complexes can yield valuable information that is often not easily available by other methods. In this article we give a number of examples that were chosen to illustrate the utility of electron microscopy. Along with the strategy used are protocols that allow such experiments to be carried out. The first example employs the following strategy. Points of close proximity between nucleic acid and protein within a bacteriophage or virus are made permanent by crosslinking. Bacteriophage or virus are then partially disrupted so that individual components can be visualized. With bacteriophages, such experiments show which DNA end first enters the host on infection and therefore can in principle indicate which phage genes would be first available for transcription. This type of experiment can also show which DNA end is first to be encapsulated during formation of the bacteriophage. Information on direction of encapsulation and indirectly, direction of replication of the rolling circles that lead to concatermeric DNA to be encapsulated, can also be derived. Such experiments can additionally accurately define the degree of DNA permutation, if present, within a bacteriophage population. Finally, examples are shown for in vitro reactions involving DNA, RecA, RecO, RecF, RecR, and SSB that lead to a further understanding of recombinational repair. Additionally antibody-gold labeling is used to locate various proteins in such complexes.

Complete nucleotide sequence, molecular analysis and genome structure of bacteriophage A118 of Listeria monocytogenes: implications for phage evolution.

A118 is a temperate phage isolated from Listeria monocytogenes. In this study, we report the entire nucleotide sequence and structural analysis of its 40 834 bp DNA. Electron microscopic and enzymatic analyses revealed that the A118 genome is a linear, circularly permuted, terminally redundant collection of double-stranded DNA molecules. No evidence for cohesive ends or for a terminase recognition (pac) site could be obtained, suggesting that A118 viral DNA is packaged via a headful mechanism. Partial denaturation mapping of DNA cross-linked to the tail shaft indicated that DNA packaging proceeds from left to right with respect to the arbitrary genomic map and the direction of genes necessary for lytic development. Seventy-two open reading frames (ORFs) were identified on the A118 genome, which are apparently organized in a life cycle-specific manner into at least three major transcriptional units. N-terminal amino acid sequencing, bioinformatic analyses and functional characterizations enabled the assignment of possible functions to 26 ORFs, which included DNA packaging proteins, morphopoetic proteins, lysis components, lysogeny control-associated functions and proteins necessary for DNA recombination, modification and replication. Comparative analysis of the A118 genome structure with other bacteriophages revealed local, but sometimes extensive, similarities to a number of phages spanning a broader phylogenetic range of various low G+C host bacteria, which implies relatively recent exchange of genes or genetic modules. We have also identified the A118 attachment site attP and the corresponding attB in Listeria monocytogenes, and show that site-specific integration of the A118 prophage by the A118 integrase occurs into a host gene homologous to comK of Bacillus subtilis, an autoregulatory gene specifying the major competence transcription factor.

ATP hydrolysis and DNA binding by the Escherichia coli RecF protein.

The Escherichia coli RecF protein possesses a weak ATP hydrolytic activity. ATP hydrolysis leads to RecF dissociation from double-stranded (ds)DNA. The RecF protein is subject to precipitation and an accompanying inactivation in vitro when not bound to DNA. A mutant RecF protein that can bind but cannot hydrolyze ATP (RecF K36R) does not readily dissociate from dsDNA in the presence of ATP. This is in contrast to the limited dsDNA binding observed for wild-type RecF protein in the presence of ATP but is similar to dsDNA binding by wild-type RecF binding in the presence of the nonhydrolyzable ATP analog, adenosine 5'-O-(3-thio)triphosphate (ATPgammaS). In addition, wild-type RecF protein binds tightly to dsDNA in the presence of ATP at low pH where its ATPase activity is blocked. A transfer of RecF protein from labeled to unlabeled dsDNA is observed in the presence of ATP but not ATPgammaS. The transfer is slowed considerably when the RecR protein is also present. In competition experiments, RecF protein appears to bind at random locations on dsDNA and exhibits no special affinity for single strand/double strand junctions when bound to gapped DNA. Possible roles for the ATPase activity of RecF in the regulation of recombinational DNA repair are discussed.

Assemblies of replication initiator protein on symmetric and asymmetric DNA sequences depend on multiple protein oligomerization surfaces.

The pi35.0 protein of plasmid R6K regulates transcription and replication by binding a DNA sequence motif (TGAGR) arranged either asymmetrically into 22 bp direct repeats (DRs) in the gamma origin, or symmetrically into inverted half-repeats (IRs) in the operator of its own gene, pir. The binding patterns of the two natural forms of the pi protein and their heterodimers revealed that the predominant species, pi35.0 (35.0 kDa), can bind to a single copy of the DR as either a monomer or a dimer while pi30.5 (30.5 kDa) binds only as a dimer. We demonstrate that only one subunit of a pi35.0 dimer makes specific contact with DNA. Electron microscopic (EM) analysis of the nucleoprotein complexes formed by pi35.0 and DNA fragments containing all seven DRs revealed coupled ("hand-cuffed") DNA molecules that are aligned in a parallel orientation. Antiparallel orientations of the DNA were not observed. Thus, hand-cuffing depends on a highly ordered oligomerization of pi35.0 in such structures. The pi protein (pi35.0, pi30.5) binds to an IR as a dimer or heterodimer but not as a monomer. Moreover, a single amino acid residue substitution, F200S (pir200), introduced into pi30.5 severely destabilizes dimers of this protein in solution and concomitantly prevents binding of this protein to the IR. This mutation also changes the stability of pi35.0 dimers but it does not change the ability of pi35.0 to bind IRs. To explain these observations we propose that the diverse interactions of pi variants with DNA are controlled by multiple surfaces for protein oligomerization.

Replication of R6K gamma origin in vitro: discrete start sites for DNA synthesis dependent on pi and its copy-up variants.

The regulation of the plasmid R6K gamma origin (gamma ori) is accomplished through the ability of the pi protein to act as an initiator and inhibitor of replication. Hyperactive variants of this protein, called copy-up pi, allow four to tenfold increases of gamma ori plasmid DNA in vivo. The higher activity of copy-up pi variants could be explained by an increase in the initiator function, a decrease in the inhibitor activity, or a derepression of a more efficient mechanism of replication that can be used by wt pi (pi35. 0) only under certain conditions. We have compared the replication activities of wt pi35.0 and copy-up pi mutants in vitro, and analyzed the replication products. It is shown that copy-up variants are several-fold more active than wt pi35.0 in replication. This appears to be due to enhanced specific replication activity of copy-up mutants rather than elevated fractions of protein proficient in DNA binding. Furthermore, biochemical complementation revealed that pi200 (copy-up) is dominant over wt pi35.0. The elevated activity of copy-up pi is not caused by an increased rate of replisome assembly as inferred from in vitro replication assays in which the lag periods observed were similar to that of wt pi35.0. Moreover, only one round of semiconservative, unidirectional replication occurred in all the samples analyzed indicating that copy-up pi proteins do not initiate multiple rounds of DNA synthesis. Rather, a larger fraction of DNA template replicates in the presence of copy-up pi as determined by electron microscopy. Two clusters of discrete DNA synthesis start sites are mapped by primer extension near the stability (stb) locus of the gamma ori. We show that the start sites are the same in the presence of wt pi35.0 or copy-up proteins. This comparative analysis suggests that wt pi35.0 and copy-up variants utilize fundamentally similar mechanism(s) of replication priming.

Recombinational DNA repair: the RecF and RecR proteins limit the extension of RecA filaments beyond single-strand DNA gaps.

In the presence of both the RecF and RecR proteins, RecA filament extension from a single strand gap into adjoining duplex DNA is attenuated. RecR protein alone has no effect, and RecF protein alone has a reduced activity. The RecFR complexes bind randomly, primarily to the duplex regions of the DNA, and the extension of the RecA filament is halted at the first complex encountered. A very slow lengthening of RecA filaments observed in the presence of RecFR is virtually eliminated when RecF is replaced with an RecF mutant protein that does not hydrolyze ATP. These observations are incorporated into an expanded model for the functions of RecF, RecO, and RecR proteins in the early stages of postreplication DNA repair.

RecA as a motor protein. Testing models for the role of ATP hydrolysis in DNA strand exchange.

ATP hydrolysis (by RecA protein) fundamentally alters the properties of RecA protein-mediated DNA strand exchange reactions. ATP hydrolysis renders DNA strand exchange unidirectional, greatly increases the lengths of hybrid DNA created, permits the bypass of heterologous DNA insertions in one or both DNA substrates, and is absolutely required for exchange reactions involving four DNA strands. There are at least two viable models to explain how ATP hydrolysis is coupled to DNA strand exchange so as to bring about these effects. The first couples ATP hydrolysis to a redistribution of RecA monomers within a RecA filament. The second couples ATP hydrolysis to a facilitated rotation of the DNA substrates. The RecA monomer redistribution model makes the prediction that heterology bypass should not occur if the single-stranded DNA substrate is linear. The facilitated DNA rotation model predicts that RecA protein should promote the separation of paired DNA strands within a RecA filament if one of them is contiguous with a length of DNA being rotated about the filament exterior. Here, a facile bypass of heterologous insertions with linear DNA substrates is demonstrated, providing evidence against a role for RecA monomer redistribution in heterology bypass. In addition, we demonstrate that following a four-strand DNA exchange reaction, a distal segment of DNA hundreds of base pairs in length can be unwound in a nonreciprocal phase of the reaction, consistent with the direct coupling of an ATP hydrolytic motor to the proposed DNA rotation.

Replication of the R6K gamma origin in vitro: dependence on wt pi and hyperactive piS87N protein variant.

The pi protein of plasmid R6K is involved in control of replication. The aim of this study was to use an in vitro replication system dependent on an R6K-derived gamma origin of replication (gamma ori) to compare replication characteristics of wt pi and a hyperactive variant of pi protein (piS87N; Filutowicz et al., 1994b. Cooperative binding of initiator protein to replication origin conferred by single amino acid substitution. Nucleic Acids Res. 22, 4211-4215). The characteristics of in vitro replication from gamma ori reported in this investigation are as follows: (i) piS87N is considerably more active in comparison to wt pi. (ii) Replication proceeds through Cairns-type intermediates and the initiation site and directionality of the fork movement are similar in the presence of both proteins. (iii) Replication forks emanate unidirectionally in the vicinity of the cluster of seven 22-bp direct repeats within gamma ori. (iv) Replication dependent on wt pi, but not piS87N, is stimulated up to 1.5-fold by rifampicin.

RecA protein filaments: end-dependent dissociation from ssDNA and stabilization by RecO and RecR proteins.

RecA protein filaments formed on circular (ssDNA) in the presence of ssDNA binding protein (SSB) are generally stable as long as ATP is regenerated. On linear ssDNA, stable RecA filaments are believed to be formed by nucleation at random sites on the DNA followed by filament extension in the 5' to 3' direction. This view must now be enlarged as we demonstrate that RecA filaments formed on linear ssDNA are subject to a previously undetected end-dependent disassembly process. RecA protein slowly dissociates from one filament end and is replaced by SSB. The results are most consistent with disassembly from the filament end nearest the 5' end of the DNA. The bound SSB prevents re-formation of the RecA filaments, rendering the dissociation largely irreversible. The dissociation requires ATP hydrolysis. Disassembly is not observed when the pH is lowered to 6.3 or when dATP replaces ATP. Disassembly is not observed even with ATP when both the RecO and RecR proteins are present in the initial reaction mixture. When the RecO and RecR proteins are added after most of the RecA protein has already dissociated, RecA protein filaments re-form after a short lag. The newly formed filaments contain an amount of RecA protein and exhibit an ATP hydrolysis rate comparable to that observed when the RecO and RecR proteins are included in the initial reaction mixture. The RecO and RecR proteins thereby stabilize RecA filaments even at the 5' ends of ssDNA, a fact which should affect the recombination potential of 5' ends relative to 3' ends. The location and length of RecA filaments involved in recombinational DNA repair is dictated by both the assembly and disassembly processes, as well as by the presence or absence of a variety of other proteins that can modulate either process.

DNA strand exchange promoted by RecA K72R. Two reaction phases with different Mg2+ requirements.

Replacement of lysine 72 in RecA protein with arginine produces a mutant protein that binds but does not hydrolyze ATP. The protein nevertheless promotes DNA strand exchange (Rehrauer, W. M., and Kowalczykowski, S. C. (1993) J. Biol. Chem. 268, 1292-1297). With RecA K72R protein, the formation of the hybrid DNA product of strand exchange is greatly affected by the concentration of Mg2+ in ways that reflect the concentration of a Mg.dATP complex. When Mg2+ is present at concentrations just sufficient to form the Mg.dATP complex, substantial generation of completed product hybrid DNAs over 7 kilobase pairs in length is observed (albeit slowly). Higher levels of Mg2+ are required for optimal uptake of substrate duplex DNA into the nucleoprotein filament, indicating that the formation of joint molecules is facilitated by Mg2+ levels that inhibit the subsequent migration of a DNA branch. We also show that the strand exchange reaction promoted by RecA K72R, regardless of the Mg2+ concentration, is bidirectional and incapable of bypassing structural barriers in the DNA or accommodating four DNA strands. The reaction exhibits the same limitations as that promoted by wild type RecA protein in the presence of adenosine 5'-O-(3-thio)triphosphate. The Mg2+ effects, the limitations of RecA-mediated DNA strand exchange in the absence of ATP hydrolysis, and unusual DNA structures observed by electron microscopy in some experiments, are interpreted in the context of a model in which a fast phase of DNA strand exchange produces a discontinuous three-stranded DNA pairing intermediate, followed by a slow phase in which the discontinuities are resolved. The mutant protein also facilitates the autocatalytic cleavage of the LexA repressor, but at a reduced rate.

An interaction between the Escherichia coli RecF and RecR proteins dependent on ATP and double-stranded DNA.

The DNA binding and ATPase activities of RecF protein are modulated by RecR protein. Stoichiometric amounts of RecF protein bind to double-stranded (ds) DNA (about 1 RecF monomer/4-6 base pairs) in the presence of adenosine 5'-O-(3-thio)triphosphate (ATP gamma S), forming a homogeneous protein coating on the DNA. Little or no cooperativity is evident in the binding process. In the presence of ATP, RecF binding to dsDNA is much weaker, and no RecF protein coating forms. Instead, small numbers of RecF protomers are interspersed randomly along the DNA. RecR protein does not bind appreciably to the dsDNA under these same conditions. However, a protein coating, similar to that which was observed with RecF protein alone in the presence of ATP gamma S, was produced when both RecF and RecR proteins were incubated with dsDNA in the presence of ATP. An interaction between RecF and RecR enables both proteins to bind tightly to the dsDNA in an approximately 1:1 molar ratio. We also report a weak ATP hydrolytic activity of RecF which is stimulated by RecR.

Blocked RecA protein-mediated DNA strand exchange reactions are reversed by the RuvA and RuvB proteins.

RecA protein is unable to complete a DNA strand exchange reaction between a circular single-stranded DNA and a linear duplex DNA substrate with heterologous sequences of 375 base pairs at the distal end. Instead, it generates a branched intermediate in which strand exchange has proceeded up to the homology/heterology junction. Addition of the RuvA and RuvB proteins to these stalled intermediates leads to the rapid conversion of intermediates back to the original substrates. The reversal reaction is initiated at the branch, and the hybrid DNA is unwound in the direction opposite to that of the RecA reaction that created it. Under optimal conditions the rate of the reaction exhibits only a modest dependence on the length of hybrid DNA that must be unwound. Products of the reversal reaction are detected within minutes after addition of RuvAB, and appear with an apparent first order progress curve, exhibiting a t1/2 in the range of 6-12 min under optimal conditions. Few molecules that have undergone only partial reversal are detected. This suggests that the assembly or activation of RuvAB on the branched substrate is rate-limiting, while any migration of RuvAB on the DNA to effect unwinding of the hybrid DNA (and reformation of substrate DNA) is very fast. The results are discussed in context of the role of RuvA and RuvB proteins in recombinational DNA repair. We suggest that one function of the RuvAB proteins is to act as an antirecombinase, to eliminate intragenomic crossovers between homologous segments of the bacterial chromosome that might otherwise lead to deleterious inversions or deletions.

Occurrence of three-stranded DNA within a RecA protein filament.

A RecA protein-generated triple-stranded DNA species can be observed by electron microscopy, within narrowly defined conditions. Three-stranded DNA is detected only when initiation of normal DNA strand exchange is precluded by heterologous sequences within the duplex DNA substrate, when ATP is hydrolyzed, and when the DNA is cross-linked with a psoralen derivative prior to removal of RecA filaments. When adenosine 5'-O-(thiotriphosphate) is used, only the product hybrid duplex DNA can be cross-linked within the RecA filament. The third strand is either displaced or interwound in a conformation that does not permit cross-linking. When ATP is hydrolyzed by RecA, all three strands are cross-linked within the filament in a complex pattern that suggests a dynamic structure. This structure is altered when RecA protein is removed before cross-linking. Hsieh et al. (1990) and Rao et al. (1991, 1993) have proposed, on the basis of nuclease protection and chemical modification studies, that a stable triple-stranded DNA species can persist after removal of RecA protein. We have been unable to visualize these triple-stranded structures by the methods used in the present investigation. When RecA removal was followed immediately by interstrand cross-linking, only the two strands of the hybrid duplex DNA were cross-linked.

Efficient concerted integration of retrovirus-like DNA in vitro by avian myeloblastosis virus integrase.

We report the efficient concerted integration of a linear virus-like DNA donor into a 2.8 kbp circular DNA target by integrase (IN) purified from avian myeloblastosis virus. The donor was 528 bp, contained recessed 3' OH ends, was 5' end labeled, and had a unique restriction site not found in the target. Analysis of concerted (full-site) and half-site integration events was accomplished by restriction enzyme analysis and agarose gel electrophoresis. The donor also contained the SupF gene that was used for genetic selection of individual full-site recombinants to determine the host duplication size. Two different pathways, involving either one donor or two donor molecules, were used to produce full-site recombinants. About 90% of the full-site recombinants were the result of using two donor molecules per target. These results imply that juxtapositioning an end from each of two donors by IN was more efficient than the juxtapositioning of two ends of a single donor for the full-site reaction. The formation of preintegration complexes containing integrase and donor on ice prior to the addition of target enhanced the full-site reaction. After a 30 min reaction at 37 degrees C, approximately 20-25% of all donor/target recombinants were the result of concerted integration events. The efficient production of full-site recombinants required Mg2+; Mn2+ was only efficient for the production of half-site recombinants. We suggest that these preintegration complexes can be used to investigate the relationships between the 3' OH trimming and strand transfer reactions.

RuvA and RuvB proteins facilitate the bypass of heterologous DNA insertions during RecA protein-mediated DNA strand exchange.

RecA protein-mediated DNA strand exchange between circular single-stranded DNA and linear duplex DNA readily bypasses short (up to 100 base pairs) heterologous inserts in one of the DNA substrates. Larger heterologous inserts are bypassed with decreasing efficiency, and inserts larger than 200 base pairs substantially block RecA-mediated DNA strand exchange. The RuvA and RuvB proteins dramatically facilitate the bypass of larger heterologous inserts. When the RuvA and RuvB proteins are added to an ongoing RecA protein-mediated strand exchange reaction, interior heterologous inserts of 1 kilobase pair are bypassed at significant frequencies. The RuvA, RuvB, and RecA proteins are all required for this activity. Bypass occurs only when homologous sequences are present on both sides of the insert. When the heterologous insert is positioned at either end of the linear duplex substrate, the RuvA and RuvB proteins do not significantly increase product formation in RecA protein-mediated DNA strand exchange reactions. The results suggest an important role for RuvA and RuvB in the bypass of DNA structural barriers during recombinational DNA repair.

On the role of ATP hydrolysis in RecA protein-mediated DNA strand exchange. III. Unidirectional branch migration and extensive hybrid DNA formation.

We have identified two functions for RecA-mediated ATP hydrolysis during DNA strand exchange. First, ATP hydrolysis renders RecA protein-mediated DNA strand exchange unidirectional (5' to 3' with respect to the single-stranded DNA). In the presence of a nonhydrolyzable analog adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S), DNA strand exchange is bidirectional. Second, ATP hydrolysis is required for extensive formation of hybrid DNA. In the presence of ATP hydrolysis, the length of the exchanged region is limited only by the available homology, whereas in the absence of ATP hydrolysis, only 2 kilobase pairs or less of hybrid DNA are formed before branch migration is blocked in the majority of paired intermediates. Both of these functions of RecA protein-mediated ATP hydrolysis are crucial in ensuring the effectiveness of recombinational DNA repair, especially when the lesion to be repaired is distant from the initial crossover point.