RecA, Tus protein and constitutive stable DNA replication inEscherichia coli rnhA mutants

Constitutive stable DNA replication (cSDR), which uniquely occurs inEscherichia coli rnhA mutants deficient in ribonuclease HI activity, requires RecA function. TherecA428 mutation, which inactivates the recombinase activity but imparts a constitutive coprotease activity, blocks cSDR inrnhA mutants. The result indicates that the recombinase activity of RecA, which promotes homologous pairing and strand exchange, is essential for cSDR. Despite the requirement for RecA recombinase activity, mutations inrecB, recD, recJ, ruvA andruvC neither inhibit nor stimulate cSDR. It was proposed that the property of RecA essential for homologous pairing and strand exchange is uniquely required for initiation of cSDR inrnhA mutants without involving the homologous recombination process. The possibility that RecA protein is necessary to counteract the action of Tus protein, a contra-helicase which stalls replication forks in theter region of the chromosome, was ruled out because introduction of thetus : :kan mutation, which inactivates Tus protein, did not alleviate the RecA requirement for cSDR.     

Constitutive stable DNA replication in Escherichia coli cells lacking type 1A topoisomerase activity

Type 1A topoisomerases (topos) are ubiquitous enzymes involved in supercoiling regulation and in the maintenance of genome stability. Escherichia coli possesses two type 1A enzymes, topo I (topA) and topo III (topB). Cells lacking both enzymes form very long filaments and have severe chromosome segregation and growth defects. We previously found that RNase HI overproduction or a dnaT::aph mutation could significantly correct these phenotypes. This leads us to hypothesize that they were related to unregulated replication originating from R-loops, i.e. constitutive stable DNA replication (cSDR). cSDR, first observed in rnhA (RNase HI) mutants, is characterized by its persistence for several hours following protein synthesis inhibition and by its requirement for primosome components, including DnaT. Here, to visualize and measure cSDR, the incorporation of the nucleotide analog ethynyl deoxyuridine (EdU) during replication in E. coli cells pre-treated with protein synthesis inhibitors, was revealed by “click” labeling with Alexa Fluor^® 488 in fixed cells, and flow cytometry analysis. cSDR was detected in rnhA mutants, but not in wild-type strains, and the number of cells undergoing cSDR was significantly reduced by the introduction of the dnaT::aph mutation. cSDR was also found in topA, double topA topB but not in topB null cells. This result is consistent with the established function of topo I in the inhibition of R-loop formation. Moreover, our finding that topB rnhA mutants are perfectly viable demonstrates that topo III is not uniquely required during cSDR. Thus, either topo I or III can provide the type 1A topo activity that is specifically required during cSDR to allow chromosome segregation.     

Disassembly of Escherichia coli RecA E38K/��C17 Nucleoprotein Filaments Is Required to Complete DNA Strand Exchange

Disassembly of RecA protein subunits from a RecA filament has long been known to occur during DNA strand exchange, although its importance to this process has been controversial. An Escherichia coli RecA E38K/ΔC17 double mutant protein displays a unique and pH-dependent mutational separation of DNA pairing and extended DNA strand exchange. Single strand DNA-dependent ATP hydrolysis is catalyzed by this mutant protein nearly normally from pH 6 to 8.5. It will also form filaments on DNA and promote DNA pairing. However, below pH 7.3, ATP hydrolysis is completely uncoupled from extended DNA strand exchange. The products of extended DNA strand exchange do not form. At the lower pH values, disassembly of RecA E38K/ΔC17 filaments is strongly suppressed, even when homologous DNAs are paired and available for extended DNA strand exchange. Disassembly of RecA E38K/ΔC17 filaments improves at pH 8.5, whereas complete DNA strand exchange is also restored. Under these sets of conditions, a tight correlation between filament disassembly and completion of DNA strand exchange is observed. This correlation provides evidence that RecA filament disassembly plays a major role in, and may be required for, DNA strand exchange. A requirement for RecA filament disassembly in DNA strand exchange has a variety of ramifications for the current models linking ATP hydrolysis to DNA strand exchange.     

RadD is a RecA-dependent accessory protein that accelerates DNA strand exchange

In rapidly growing cells, with recombinational DNA repair required often and a new replication fork passing every 20 min, the pace of RecA-mediated DNA strand exchange is potentially much too slow for bacterial DNA metabolism. The enigmatic RadD protein, a putative SF2 family helicase, exhibits no independent helicase activity on branched DNAs. Instead, RadD greatly accelerates RecA-mediated DNA strand exchange, functioning only when RecA protein is present. The RadD reaction requires the RadD ATPase activity, does not require an interaction with SSB, and may disassemble RecA filaments as it functions. We present RadD as a new class of enzyme, an accessory protein that accelerates DNA strand exchange, possibly with a helicase-like action, in a reaction that is entirely RecA-dependent. RadD is thus a DNA strand exchange (recombination) synergist whose primary function is to coordinate closely with and accelerate the DNA strand exchange reactions promoted by the RecA recombinase. Multiple observations indicate a uniquely close coordination of RadD with RecA function.     

RecA-mediated strand exchange traverses substitutional heterologies more easily than deletions or insertions

RecA protein in bacteria and its eukaryotic homolog Rad51 protein are responsible for initiation of strand exchange between homologous DNA molecules. This process is crucial for homologous recombination, the repair of certain types of DNA damage and for the reinitiation of DNA replication on collapsed replication forks. We show here, using two different types of in vitro assays, that in the absence of ATP hydrolysis RecA-mediated strand exchange traverses small substitutional heterologies between the interacting DNAs, whereas small deletions or insertions block the ongoing strand exchange. We discuss evolutionary implications of RecA selectivity against insertions and deletions and propose a molecular mechanism by which RecA can exert this selectivity.     

Requirement for the polymerization and 5'-->3' exonuclease activities of DNA polymerase I in initiation of DNA replication at oriK sites in the absence of RecA in Escherichia coli rnhA mutants.

In previous studies, we found that the requirement for RecA protein in constitutive stable DNA replication (cSDR) can be bypassed by derepression of the LexA regulon and that DNA polymerase I (DNA PolI) is essential for this Rip (RecA-independent process) pathway of cSDR (Y. Cao, R. R. Rowland, and T. Kogoma, J. Bacteriol. 175:7247-7253, 1993). In this study, the role of DNA PolI in the Rip pathway was further examined. By using F' plasmids carrying different parts of the polA gene, a series of complementation tests was carried out to investigate the requirement for the three enzymatic activities, polymerization, 3'-->5' exonuclease, and 5'-->3' exonuclease activities, of DNA PolI. The result indicated that both the 5'-->3' exonuclease and polymerization activities of DNA PolI are essential for bypassing the requirement for RecA in cSDR but that the 3'-->5' exonuclease activity can be dispensed with. Complementation experiments with rat DNA Pol beta also supported the hypothesis that a nick translation activity is probably involved in cSDR in the absence of RecA. An analysis of DNA synthesis suggested that DNA PolI is involved in the initiation but not the elongation stage of cSDR. Moreover, the dnaE293(Ts) mutation was shown to render the bypass replication temperature sensitive despite the presence of active DNA PolI, suggesting that DNA PolIII is responsible for the elongation stage of the Rip pathway. A model which describes the possible roles of RecA in cSDR and the possible function of DNA PolI in the Rip pathway is proposed.     

Magnesium ion-dependent activation of the RecA protein involves the C terminus

Optimal conditions for RecA protein-mediated DNA strand exchange include 6-8 mm Mg(2+) in excess of that required to form complexes with ATP. We provide evidence that the free magnesium ion is required to mediate a conformational change in the RecA protein C terminus that activates RecA-mediated DNA strand exchange. In particular, a "closed" (low Mg(2+)) conformation of a RecA nucleoprotein filament restricts DNA pairing by incoming duplex DNA, although single-stranded overhangs at the ends of a duplex allow limited DNA pairing to occur. The addition of excess Mg(2+) results in an "open" conformation, which can promote efficient DNA pairing and strand exchange regardless of DNA end structure. The removal of 17 amino acid residues at the Escherichia coli RecA C terminus eliminates a measurable requirement for excess Mg(2+) and permits efficient DNA pairing and exchange similar to that seen with the wild-type protein at high Mg(2+) levels. Thus, the RecA C terminus imposes the need for the high magnesium ion concentrations requisite in RecA reactions in vitro. We propose that the C terminus acts as a regulatory switch, modulating the access of double-stranded DNA to the presynaptic filament and thereby inhibiting homologous DNA pairing and strand exchange at low magnesium ion concentrations.     

Molecular visualization of the yeast Dmc1 protein ring and Dmc1-ssDNA nucleoprotein complex

Saccharomyces cerevisiae Dmc1, a meiosis-specific homologue of RecA, catalyzes homologous pairing and strand exchange during meiotic DNA recombination. The purified budding yeast Dmc1 (ScDmc1) protein exhibits much weaker recombinase activity in vitro as compared to that of the Escherichia coli RecA protein. Using atomic force microscopy (AFM) with carbon nanotube tips, we found ScDmc1 forms rings with an external diameter of 18 nm and a central cavity of 4 nm. In the presence of single-stranded DNA (ssDNA), the majority of the ScDmc1 protein (90%) bound DNA as protein rings; only a small faction (10%) was able to form filamentous structure. In contrast, nearly all RecA proteins form fine helical nucleoprotein filaments with ssDNA under identical conditions. RecA-mediated recombinase activity is initiated through the nucleation of RecA onto ssDNA to form helical nucleoprotein filaments. Our results support the notion that ScDmc1 becomes catalytically active only when it forms a helical nucleoprotein filament with ssDNA.     

Tus-mediated arrest of DNA replication in Escherichia coli is modulated by DNA supercoiling

In the absence of RecA, expression of the Tus protein of Escherichia coli is lethal when ectopic Ter sites are inserted into the chromosome in an orientation that blocks completion of chromosome replication. Using this observation as a basis for genetic selection, an extragenic suppressor of Tus-mediated arrest of DNA replication was isolated with diminished ability of Tus to halt DNA replication. Resistance to tus expression mapped to a mutation in the stop codon of the topA gene (topA869), generating an elongated topoisomerase I protein with a marked reduction in activity. Other alleles of topA with mutations in the carboxyl-terminal domain of topoisomerase I, topA10 and topA66, also rendered recA strains with blocking Ter sites insensitive to tus expression. Thus, increased negative supercoiling in the DNA of these mutants reduced the ability of Tus-Ter complexes to arrest DNA replication. The increase in superhelical density did not diminish replication arrest by disrupting Tus-Ter interactions, as Tus binding to Ter sites was essentially unaffected by the topA mutations. The topA869 mutation also relieved the requirement for recombination functions other than recA to restart replication, such as recC, ruvA and ruvC, indicating that the primary effect of the increased negative supercoiling was to interfere with Tus blockage of DNA replication. Introduction of gyrB mutations in combination with the topA869 mutation restored supercoiling density to normal values and also restored replication arrest at Ter sites, suggesting that supercoiling alone modulated Tus activity. We propose that increased negative supercoiling enhances DnaB unwinding activity, thereby reducing the duration of the Tus-DnaB interaction and leading to decreased Tus activity.     

Developing Single-Molecule TPM Experiments for Direct Observation of Successful RecA-Mediated Strand Exchange Reaction

RecA recombinases play a central role in homologous recombination. Once assembled on single-stranded (ss) DNA, RecA nucleoprotein filaments mediate the pairing of homologous DNA sequences and strand exchange processes. We have designed two experiments based on tethered particle motion (TPM) to investigate the fates of the invading and the outgoing strands during E. coli RecA-mediated pairing and strand exchange at the single-molecule level in the absence of force. TPM experiments measure the tethered bead Brownian motion indicative of the DNA tether length change resulting from RecA binding and dissociation. Experiments with beads labeled on either the invading strand or the outgoing strand showed that DNA pairing and strand exchange occurs successfully in the presence of either ATP or its non-hydrolyzable analog, ATPγS. The strand exchange rates and efficiencies are similar under both ATP and ATPγS conditions. In addition, the Brownian motion time-courses suggest that the strand exchange process progresses uni-directionally in the 5′-to-3′ fashion, using a synapse segment with a wide and continuous size distribution.     

Entamoeba histolytica Dmc1 Catalyzes Homologous DNA Pairing and Strand Exchange That Is Stimulated by Calcium and Hop2-Mnd1

Meiosis depends on homologous recombination (HR) in most sexually reproducing organisms. Efficient meiotic HR requires the activity of the meiosis-specific recombinase, Dmc1. Previous work shows Dmc1 is expressed in Entamoeba histolytica, a eukaryotic parasite responsible for amoebiasis throughout the world, suggesting this organism undergoes meiosis. Here, we demonstrate Dmc1 protein is expressed in E. histolytica. We show that purified ehDmc1 forms presynaptic filaments and catalyzes ATP-dependent homologous DNA pairing and DNA strand exchange over at least several thousand base pairs. The DNA pairing and strand exchange activities are enhanced by the presence of calcium and the meiosis-specific recombination accessory factor, Hop2-Mnd1. In combination, calcium and Hop2-Mnd1 dramatically increase the rate of DNA strand exchange activity of ehDmc1. The biochemical system described herein provides a basis on which to better understand the role of ehDmc1 and other HR proteins in E. histolytica.     

ADP-dependent DNA strand exchange by the mutant [P67G/E68A] RecA protein

We have prepared a mutant RecA protein in which proline 67 and glutamic acid 68 in the NTP binding site were replaced by a glycine and alanine residue, respectively. The [P67G/E68A]RecA protein catalyzes the single-stranded DNA-dependent hydrolysis of ATP and is able to promote the standard ATP-dependent three-strand exchange reaction between a circular bacteriophage phiX174 (phiX) single-stranded DNA molecule and a homologous linear phiX double-stranded (ds) DNA molecule (5.4 kilobase pairs). The strand exchange activity differs from that of the wild type RecA protein, however, in that it is (i) completely inhibited by an ATP regeneration system, and (ii) strongly stimulated by the addition of high concentrations of ADP to the reaction solution. These results indicate that the strand exchange activity of the [P67G/E68A]RecA protein is dependent on the presence of both ATP and ADP. The ADP dependence of the reaction is reduced or eliminated when (i) a shorter linear phiX dsDNA fragment (1.1 kilobase pairs) is substituted for the full-length linear phiX dsDNA substrate, or (ii) the Mg(2+) concentration is reduced to a level just sufficient to complex the ATP present in the reaction solution. These results indicate that it is the branch migration phase (and not the initial pairing step) of the [P67G/E68A]RecA protein-promoted strand exchange reaction that is dependent on ADP. It is likely that the [P67G/E68A]RecA mutation has revealed a requirement for ADP that also exists (but is not as readily apparent) in the strand exchange reaction of the wild type RecA protein.     

Electron microscopic visualization of the RecA protein-mediated pairing and branch migration phases of DNA strand exchange

The RecA protein of Escherichia coli will drive the pairing and exchange of strands between homologous DNA molecules in a reaction stimulated by single-stranded binding protein. Here, reactions utilizing three homologous DNA pairs which can undergo both paranemic and plectonemic joining were examined by electron microscopy: supertwisted double-stranded (ds) DNA and linear single-stranded (ss) DNA, linear dsDNA and circular ssDNA, and linear dsDNA and colinear ssDNA. Several major observations were: (i) with RecA protein bound to the DNA, plectonemic joints were ultrastructurally indistinguishable from paranemic joints; (ii) complexes which appeared to be joined both paranemically and plectonemically were present in these reactions in roughly equal numbers; and (iii) in complexes undergoing strand exchange, both DNA partners were often enveloped within a RecA protein filament consisting of hundreds of RecA protein monomers and several kilobases of DNA. These observations suggest that, following RecA protein-ssDNA filament formation, strand exchange proceeds by a pathway that can be divided structurally into three phases: pairing, envelopment/exchange, and release of the products.     

The herpes simplex virus type-1 single-strand DNA-binding protein (ICP8) promotes strand invasion

ICP8, the herpes simplex virus type-1 single-strand DNA-binding protein, was recently shown to promote strand exchange in conjunction with the viral replicative helicase (Nimonkar, A. V., and Boehmer, P. E. (2002) J. Biol. Chem. 277, 15182-15189). Here we show that ICP8 also catalyzes strand invasion in an ATP-independent manner. Thus, ICP8 promotes the assimilation of a single-stranded donor molecule into a homologous plasmid, resulting in the formation of a displacement loop. Invasion of a homologous duplex by single-stranded DNA requires homology at either 3' or 5' end of the invading strand. The reaction is dependent on the free energy of supercoiling and alters the topology of the acceptor plasmid. Hence, strand invasion products formed by ICP8 are resistant to the action of restriction endonucleases that cleave outside of the area of pairing. The ability to catalyze strand invasion is a novel activity of ICP8 and the first demonstration of a eukaryotic viral single-strand DNA-binding protein to promote this reaction. In this regard ICP8 is functionally similar to the prototypical prokaryotic recombinase RecA and its eukaryotic homologs. This strand invasion activity of ICP8 coupled with DNA synthesis may explain the high prevalence of branched DNA structures during viral replication.     

TheisfA mutation inhibits mutator activity and processing of UmuD protein inEscherichia coli recA730 strains

Further studies on theisfA mutation responsible for anti-SOS and antimutagenic activities inEscherichia coli are described. We have previously shown that theisfA mutation inhibits mutagenesis and other SOS-dependent phenomena, possibly by interfering with RecA coprotease activity. TheisfA mutation has now been demonstrated also to suppress mutator activity inE. coli recA730 andrecA730 lexA51(Def) strains that constitutively express RecA coprotease activity. We further show that the antimutator activity of theisfA mutation is related to inhibition of RecA coprotease-dependent processing of UmuD. Expression of UmuD' from plasmid pGW2122 efficiently restores UV-induced mutagenesis in therecA730 isfA strain and partially restores its mutator activity. On the other hand, overproduction of UmuD'C proteins from pGW2123 plasmid markedly enhances UV sensitivity with no restoration of mutability.     

Escherichia coli PriA protein is essential for inducible and constitutive stable DNA replication.

Under certain conditions, Escherichia coli cells exhibit either of two altered modes of chromosomal DNA replication. These are inducible stable DNA replication (iSDR), seen in SOS-induced cells, and constitutive stable DNA replication (cSDR), seen in rnhA mutants. Both iSDR and cSDR can continue to occur in the absence of protein synthesis. They are dependent on RecA protein, but do not require DnaA protein or the oriC site. Here we report the requirement for PriA, a protein essential for assembly of the phi X174-type primosome, for both iSDR and cSDR. In priA1(Null)::kan mutant cells, iSDR is not observed after induction by thymine starvation. Replication from one of the origins (oriM1) specific to iSDR is greatly reduced by the priA1::kan mutation. cSDR in rnhA224 mutant cells deficient in RNase HI is also completely abolished by the same priA mutation. In both cases, SDR is restored by introduction of a plasmid carrying a wild-type priA gene. Furthermore, the viability of an rnhA::cat dnaA46 strain is lost at 42 degrees C upon inactivation of the priA gene, indicating the lethal effect of priA inactivation on those cells whose viability depends on cSDR. These results demonstrate that a function of PriA protein is essential for iSDR and cSDR and suggest the involvement of the PriA-dependent phi X174-type primosome in these DnaA/oriC-independent pathways of chromosome replication. Whereas ColE1-type plasmids, known to be independent of DnaA, absolutely require PriA function for replication, DnaA-dependent plasmid replicons such as pSC101, F, R6K, Rts1 and RK2 are able to transform and to be maintained in the priA1::kan strain.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human Rad51 amino acid residues required for Rad52 binding.

The Rad51 protein, a homologue of the bacterial RecA protein, is an essential factor for both meiotic and mitotic recombination. The N-terminal domain of the human Rad51 protein (HsRad51) directly interacts with DNA. Based on a yeast two-hybrid analysis, it has been reported that the N-terminal region of the Saccharomyces cerevisiae Rad51 protein binds Rad52;S. cerevisiae Rad51 and Rad52 both activate the homologous pairing and strand exchange reactions. Here, we show that the HsRad51 N-terminal region, which corresponds to the Rad52-binding region of ScRad51, does not exhibit strong binding to the human Rad52 protein (HsRad52). To investigate its function, the C-terminal region of HsRad51 was randomly mutagenized. Although this region includes the two segments corresponding to the putative DNA-binding sites of RecA, all seven of the mutants did not decrease, but instead slightly increased, the DNA binding. In contrast, we found that some of these HsRad51 mutations significantly decreased the HsRad52 binding. Therefore, we conclude that these amino acid residues are required for the HsRad51.HsRad52 binding. HsRad52, as well as S. cerevisiae Rad52, promoted homologous pairing between ssDNA and dsDNA, and higher homologous pairing activity was observed in the presence of both HsRad51 and HsRad52 than with either HsRad51 or HsRad52 alone. The HsRad51 F259V mutation, which strongly impaired the HsRad52 binding, decreased the homologous pairing in the presence of both HsRad51 and HsRad52, without affecting the homologous pairing by HsRad51 alone. This result suggests the importance of the HsRad51.HsRad52 interaction in homologous pairing.     

RecA protein reinitiates strand exchange on isolated protein-free DNA intermediates. An ADP-resistant process

Efficient homologous pairing de novo of linear duplex DNA with a circular single strand (plus strand) coated with RecA protein requires saturation and extension of the single strand by the protein. However, strand exchange, the transfer of a strand from duplex DNA to the nucleoprotein filament, which follows homologous pairing, does not require the stable binding of RecA protein to single-stranded DNA. When RecA protein was added back to isolated protein-free DNA intermediates in the presence of sufficient ADP to inhibit strongly the binding of RecA protein to single-stranded DNA, strand exchange nonetheless resumed at the original rate and went to completion. Characterization of the protein-free DNA intermediate suggested that it has a special site or region to which RecA protein binds. Part of the nascent displaced plus strand of the deproteinized intermediate was unavailable as a cofactor for the ATPase activity of RecA protein, and about 30% resisted digestion by P1 endonuclease, which acts preferentially on single-stranded DNA. At the completion of strand exchange, when the distal 5' end of the linear minus strand had been fully incorporated into heteroduplex DNA, a nucleoprotein complex remained that contained all three strands of DNA from which the nascent displaced strand dissociated only over the next 50 to 60 minutes. Deproteinization of this intermediate yielded a complex that also contained three strands of DNA in which the nascent displaced strand was partially resistant to both Escherichia coli exonuclease I and P1 endonuclease. The deproteinized complex showed a broad melting transition between 37 degrees C and temperatures high enough to melt duplex DNA. These results show that strand exchange can be subdivided into two stages: (1) the exchange of base-pairs, which creates a new heteroduplex pair in place of a parental pair; and (2) strand separation, which is the physical displacement of the unpaired strand from the nucleoprotein filament. Between the creation of new heteroduplex DNA and the eventual separation of a third strand, there exists an unusual DNA intermediate that may contain three-stranded regions of natural DNA that are several thousand bases in length.     

RecA dimers serve as a functional unit for assembly of active nucleoprotein filaments

All RecA-like recombinase enzymes catalyze DNA strand exchange as elongated filaments on DNA. Despite numerous biochemical and structural studies of RecA and the related Rad51 and RadA proteins, the unit oligomer(s) responsible for nucleoprotein filament assembly and coordinated filament activity remains undefined. We have created a RecA fused dimer protein and show that it maintains in vivo DNA repair and LexA co-protease activities, as well as in vitro ATPase and DNA strand exchange activities. Our results support the idea that dimeric RecA is an important functional unit both for assembly of nucleoprotein filaments and for their coordinated activity during the catalysis of homologous recombination.