An SOS inhibitor that binds to free RecA protein: the PsiB protein.

The process of bacterial conjugation involves the transfer of a conjugative plasmid as a single strand. The potentially deleterious SOS response, which is normally triggered by the appearance of single-stranded DNA, is suppressed in the recipient cell by a conjugative plasmid system centered on the product of the psiB gene. The F plasmid PsiB protein inhibits all activities of the RecA protein, including DNA binding, DNA strand exchange, and LexA protein cleavage. The proteins known to negatively regulate recombinases, such as RecA or Rad51, generally work at the level of dismantling the nucleoprotein filament. However, PsiB binds to RecA protein that is free in solution. The RecA-PsiB complex impedes formation of RecA nucleoprotein filaments on DNA.

Disassembly of Escherichia coli RecA E38K/DeltaC17 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/DeltaC17 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/DeltaC17 filaments is strongly suppressed, even when homologous DNAs are paired and available for extended DNA strand exchange. Disassembly of RecA E38K/DeltaC17 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.

RecA-mediated SOS induction requires an extended filament conformation but no ATP hydrolysis.

The Escherichia coli SOS response to DNA damage is modulated by the RecA protein, a recombinase that forms an extended filament on single-stranded DNA and hydrolyzes ATP. The RecA K72R (recA2201) mutation eliminates the ATPase activity of RecA protein. The mutation also limits the capacity of RecA to form long filaments in the presence of ATP. Strains with this mutation do not undergo SOS induction in vivo. We have combined the K72R variant of RecA with another mutation, RecA E38K (recA730). In vitro, the double mutant RecA E38K/K72R (recA730,2201) mimics the K72R mutant protein in that it has no ATPase activity. The double mutant protein will form long extended filaments on ssDNA and facilitate LexA cleavage almost as well as wild-type, and do so in the presence of ATP. Unlike recA K72R, the recA E38K/K72R double mutant promotes SOS induction in vivo after UV treatment. Thus, SOS induction does not require ATP hydrolysis by the RecA protein, but does require formation of extended RecA filaments. The RecA E38K/K72R protein represents an improved reagent for studies of the function of ATP hydrolysis by RecA in vivo and in vitro.

DdrB protein, an alternative Deinococcus radiodurans SSB induced by ionizing radiation.

Deinococcus radiodurans exhibits an extraordinary resistance to the effects of exposure to ionizing radiation (IR). DdrB is one of five proteins induced to high levels in Deinococcus following extreme IR exposure and that play a demonstrable role in genome reconstitution. Although homology is limited, DdrB is a bacterial single-stranded DNA-binding protein. DdrB features a stable core with a putative OB-fold, and a C-terminal segment with properties consistent with other bacterial SSBs. In solution, the protein functions as a pentamer. The protein binds single-stranded DNA but not duplex DNA. Electron microscopy and assays with two RecA proteins provide further structural and functional identification with bacterial SSB. Overall, the results establish DdrB as the prototype of a new bacterial SSB family. Given the role of SSB as a mobilization scaffold for many processes in DNA metabolism, the induction of an alternative and quite novel SSB following irradiation has potentially broad significance for the organization of genome reconstitution functions.

Defective dissociation of a "slow" RecA mutant protein imparts an Escherichia coli growth defect.

The RecA and some related proteins possess a simple motif, called (KR)X(KR), that (in RecA) consists of two lysine residues at positions 248 and 250 at the subunit-subunit interface. This study and previous work implicate this RecA motif in the following: (a) catalyzing ATP hydrolysis in trans,(b) coordinating the ATP hydrolytic cycles of adjacent subunits, (c) governing the rate of ATP hydrolysis, and (d) coupling the ATP hydrolysis to work (in this case DNA strand exchange). The conservative K250R mutation leaves RecA nucleoprotein filament formation largely intact. However, ATP hydrolysis is slowed to less than 15% of the wild-type rate. DNA strand exchange is also slowed commensurate with the rate of ATP hydrolysis. The results reinforce the idea of a tight coupling between ATP hydrolysis and DNA strand exchange. When a plasmid-borne RecA K250R protein is expressed in a cell otherwise lacking RecA protein, the growth of the cells is severely curtailed. The slow growth defect is alleviated in cells lacking RecFOR function, suggesting that the defect reflects loading of RecA at stalled replication forks. Suppressors occur as recA gene alterations, and their properties indicate that limited dissociation by RecA K250R confers the slow growth phenotype. Overall, the results suggest that recombinational DNA repair is a common occurrence in cells. RecA protein plays a sufficiently intimate role in the bacterial cell cycle that its properties can limit the growth rate of a bacterial culture.

Microplitis demolitor bracovirus genome segments vary in abundance and are individually packaged in virions.

Polydnaviruses (PDVs) are distinguished by their unique association with parasitoid wasps and their segmented, double-stranded (ds) DNA genomes that are non-equimolar in abundance. Relatively little is actually known, however, about genome packaging or segment abundance of these viruses. Here, we conducted electron microscopy (EM) and real-time polymerase chain reaction (PCR) studies to characterize packaging and segment abundance of Microplitis demolitor bracovirus (MdBV). Like other PDVs, MdBV replicates in the ovaries of females where virions accumulate to form a suspension called calyx fluid. Wasps then inject a quantity of calyx fluid when ovipositing into hosts. The MdBV genome consists of 15 segments that range from 3.6 (segment A) to 34.3 kb (segment O). EM analysis indicated that MdBV virions contain a single nucleocapsid that encapsidates one circular DNA of variable size. We developed a semi-quantitative real-time PCR assay using SYBR Green I. This assay indicated that five (J, O, H, N and B) segments of the MdBV genome accounted for more than 60% of the viral DNAs in calyx fluid. Estimates of relative segment abundance using our real-time PCR assay were also very similar to DNA size distributions determined from micrographs. Analysis of parasitized Pseudoplusia includens larvae indicated that copy number of MdBV segments C, B and J varied between hosts but their relative abundance within a host was virtually identical to their abundance in calyx fluid. Among-tissue assays indicated that each viral segment was most abundant in hemocytes and least abundant in salivary glands. However, the relative abundance of each segment to one another was similar in all tissues. We also found no clear relationship between MdBV segment and transcript abundance in hemocytes and fat body.

Inhibition of RecA protein function by the RdgC protein from Escherichia coli.

The Escherichia coli RdgC protein is a potential negative regulator of RecA function. RdgC inhibits RecA protein-promoted DNA strand exchange, ATPase activity, and RecA-dependent LexA cleavage. The primary mechanism of RdgC inhibition appears to involve a simple competition for DNA binding sites, especially on duplex DNA. The capacity of RecA to compete with RdgC is improved by the DinI protein. RdgC protein can inhibit DNA strand exchange catalyzed by RecA nucleoprotein filaments formed on single-stranded DNA by binding to the homologous duplex DNA and thereby blocking access to that DNA by the RecA nucleoprotein filaments. RdgC protein binds to single-stranded and double-stranded DNA, and the protein can be visualized on DNA using electron microscopy. RdgC protein exists in solution as a mixture of oligomeric states in equilibrium, most likely as monomers, dimers, and tetramers. This concentration-dependent change of state appears to affect its mode of binding to DNA and its capacity to inhibit RecA. The various species differ in their capacity to inhibit RecA function.

Complementation of one RecA protein point mutation by another. Evidence for trans catalysis of ATP hydrolysis.

The RecA residues Lys248 and Glu96 are closely opposed across the RecA subunit-subunit interface in some recent models of the RecA nucleoprotein filament. The K248R and E96D single mutant proteins of the Escherichia coli RecA protein each bind to DNA and form nucleoprotein filaments but do not hydrolyze ATP or dATP. A mixture of K248R and E96D single mutant proteins restores dATP hydrolysis to 25% of the wild type rate, with maximum restoration seen when the proteins are present in a 1:1 ratio. The K248R/E96D double mutant RecA protein also hydrolyzes ATP and dATP at rates up to 10-fold higher than either single mutant, although at a reduced rate compared with the wild type protein. Thus, the K248R mutation partially complements the inactive E96D mutation and vice versa. The complementation is not sufficient to allow DNA strand exchange. The K248R and E96D mutations originate from opposite sides of the subunit-subunit interface. The functional complementation suggests that Lys248 plays a significant role in ATP hydrolysis in trans across the subunit-subunit interface in the RecA nucleoprotein filament. This could be part of a mechanism for the long range coordination of hydrolytic cycles between subunits within the RecA filament.

The RecF protein antagonizes RecX function via direct interaction.

The RecX protein inhibits RecA filament extension, leading to net filament disassembly. The RecF protein physically interacts with the RecX protein and protects RecA from the inhibitory effects of RecX. In vitro, efficient RecA filament formation onto single-stranded DNA binding protein (SSB)-coated circular single-stranded DNA (ssDNA) in the presence of RecX occurs only when all of the RecFOR proteins are present. The RecOR proteins contribute only to RecA filament nucleation onto SSB-coated single-stranded DNA and are unable to counter the inhibitory effects of RecX on RecA filaments. RecF protein uniquely supports substantial RecA filament extension in the presence of RecX. In vivo, RecF protein counters a RecX-mediated inhibition of plasmid recombination. Thus, a significant positive contribution of RecF to RecA filament assembly is to antagonize the effects of the negative modulator RecX, specifically during the extension phase.

Role of pi dimers in coupling ("handcuffing") of plasmid R6K's gamma ori iterons.

One proposed mechanism of replication inhibition in iteron-containing plasmids (ICPs) is "handcuffing," in which the coupling of origins via iteron-bound replication initiator (Rep) protein turns off origin function. In minimal R6K replicons, copy number control requires the interaction of plasmid-encoded pi protein with the seven 22-bp iterons of the gamma origin of replication. Like other related Rep proteins, pi exists as both monomers and dimers. However, the ability of pi dimers to bind iterons distinguishes R6K from most other ICPs, where only monomers have been observed to bind iterons. Here, we describe experiments to determine if monomers or dimers of pi protein are involved in the formation of handcuffed complexes. Standard ligation enhancement assays were done using pi variants with different propensities to bind iterons as monomers or dimers. Consistent with observations from several ICPs, a hyperreplicative variant (pi.P106L(wedge)F107S) exhibits deficiencies in handcuffing. Additionally, a novel dimer-biased variant of pi protein (pi.M36A(wedge)M38A), which lacks initiator function, handcuffs iteron-containing DNA more efficiently than does wild-type pi. The data suggest that pi dimers mediate handcuffing, supporting our previously proposed model of handcuffing in the gamma ori system. Thus, dimers of pi appear to possess three distinct inhibitory functions with respect to R6K replication: transcriptional autorepression of pi expression, in cis competition (for origin binding) with monomeric activator pi, and handcuffing-mediated inhibition of replication in trans.

Situational repair of replication forks: roles of RecG and RecA proteins.

Replication forks often stall or collapse when they encounter a DNA lesion. Fork regression is part of several major paths to the repair of stalled forks, allowing nonmutagenic bypass of the lesion. We have shown previously that Escherichia coli RecA protein can promote extensive regression of a forked DNA substrate that mimics a possible structure of a replication fork stalled at a leading strand lesion. Using electron microscopy and gel electrophoresis, we demonstrate that another protein, E. coli RecG helicase, promotes extensive fork regression in the same system. The RecG-catalyzed fork regression is very efficient and faster than the RecA-promoted reaction (up to 240 bp s(-1)), despite very limited processivity of the RecG protein. The reaction is dependent upon ATP hydrolysis and is stimulated by single-stranded binding protein. The RecA- and RecG-promoted reactions are not synergistic. In fact, RecG functions poorly under the conditions optimal for the RecA reaction, and vice versa. When both RecA and RecG proteins are incubated with the DNA substrate, high RecG concentrations inhibit the RecA protein-promoted fork regression. The very different reaction profiles may reflect a situational application of these proteins to the rescue of stalled replication forks in vivo.

The Rad51-dependent pairing of long DNA substrates is stabilized by replication protein A.

Rad51 protein forms nucleoprotein filaments on single-stranded DNA (ssDNA) and then pairs that DNA with the complementary strand of incoming duplex DNA. In apparent contrast with published results, we demonstrate that Rad51 protein promotes an extensive pairing of long homologous DNAs in the absence of replication protein A. This pairing exists only within the Rad51 filament; it was previously undetected because it is lost upon deproteinization. We further demonstrate that RPA has a critical postsynaptic role in DNA strand exchange, stabilizing the DNA pairing initiated by Rad51 protein. Stabilization of the Rad51-generated DNA pairing intermediates can be can occur either by binding the displaced strand with RPA or by degrading the same DNA strand using exonuclease VII. The optimal conditions for Rad51-mediated DNA strand exchange used here minimize the secondary structure in single-stranded DNA, minimizing the established presynaptic role of RPA in facilitating Rad51 filament formation. We verify that RPA has little effect on Rad51 filament formation under these conditions, assigning the dramatic stimulation of strand exchange nevertheless afforded by RPA to its postsynaptic function of removing the displaced DNA strand from Rad51 filaments.

Genome and proteome of Listeria monocytogenes phage PSA: an unusual case for programmed + 1 translational frameshifting in structural protein synthesis.

PSA is a temperate phage isolated from Listeria monocytogenes strain Scott A. We report its complete nucleotide sequence, which consists of a linear 37 618 bp DNA featuring invariable, 3'-protruding single stranded (cohesive) ends of 10 nucleotides. The physical characteristics were confirmed by partial denaturation mapping and electron microscopy of DNA molecules. Fifty-seven open reading frames were identified on the PSA genome, which are apparently organized into three major transcriptional units, in a life cycle-specific order. Functional assignments could be made to 33 gene products, including structural proteins, lysis components, DNA packaging proteins, lysogeny control functions and replication proteins. Bioinformatics demonstrated relatedness of PSA to phages infecting lactic acid bacteria and other low G + C Gram-positives, but revealed only few similarities to Listeria phage A118. Virion proteins were analysed by amino acid sequencing and mass spectrometry, which enabled identification of major capsid and tail proteins, a tape measure and a putative portal. These analyses also revealed an unusual form of translational frameshifting, which occurs during decoding of the mRNAs specifying the two major structural proteins. Frameshifting yields different length forms of Cps (gp5) and Tsh (gp10), featuring identical N-termini but different C-termini. Matrix-assisted laser-desorption ionization mass spectrometry (MALDI-MS) and electrospray ionization mass spectrometry (ESI-MS) of tryptic peptide fragments was used to identify the modified C-termini of the longer protein species, by demonstration of specific sequences resulting from + 1 programmed translational frameshifting. A slippery sequence with overlapping proline codons near the 3' ends of both genes apparently redirects the ribosomes and initiates the recoding event. Two different cis-acting factors, a shifty stop and a pseudoknot, presumably stimulate frameshifting efficiency. PSA represents the first case of + 1 frameshifting among dsDNA phages, and appears to be the first example of a virus utilizing a 3' pseudoknot to stimulate such an event.

The DinI protein stabilizes RecA protein filaments.

When DinI is present at concentrations that are stoichiometric with those of RecA or somewhat greater, DinI has a substantial stabilizing effect on RecA filaments bound to DNA. Exchange of RecA between free and bound forms was almost entirely suppressed, and highly stable filaments were documented with several different experimental methods. DinI-mediated stabilization did not affect RecA-mediated ATP hydrolysis and LexA co-protease activities. Initiation of DNA strand exchange was affected in a DNA structure-dependent manner, whereas ongoing strand exchange was not affected. Destabilization of RecA filaments occurred as reported in earlier work but only when DinI protein was present at very high concentrations, generally superstoichiometric, relative to the RecA protein concentration. DinI did not facilitate RecA filament formation but stabilized the filaments only after they were formed. The interaction between the RecA protein and DinI was modulated by the C terminus of RecA. We discuss these results in the context of a new hypothesis for the role of DinI in the regulation of recombination and the SOS response.

A RecA filament capping mechanism for RecX protein.

The RecX protein is a potent inhibitor of RecA protein activities. RecX functions by specifically blocking the extension of RecA filaments. In vitro, this leads to a net disassembly of RecA protein from circular single-stranded DNA. Based on multiple observations, we propose that RecX has a RecA filament capping activity. This activity has predictable effects on the formation and disassembly of RecA filaments. In vivo, the RecX protein may limit the length of RecA filaments formed during recombinational DNA repair and other activities. RecX protein interacts directly with RecA protein, but appears to interact in a functionally significant manner only with RecA filaments bound to DNA.

Circadian clock protein KaiC forms ATP-dependent hexameric rings and binds DNA.

KaiC from Synechococcus elongatus PCC 7942 (KaiC) is an essential circadian clock protein in cyanobacteria. Previous sequence analyses suggested its inclusion in the RecADnaB superfamily. A characteristic of the proteins of this superfamily is that they form homohexameric complexes that bind DNA. We show here that KaiC also forms ring complexes with a central pore that can be visualized by electron microscopy. A combination of analytical ultracentrifugation and chromatographic analyses demonstrates that these complexes are hexameric. The association of KaiC molecules into hexamers depends on the presence of ATP. The KaiC sequence does not include the obvious DNA-binding motifs found in RecA or DnaB. Nevertheless, KaiC binds forked DNA substrates. These data support the inclusion of KaiC into the RecADnaB superfamily and have important implications for enzymatic activity of KaiC in the circadian clock mechanism that regulates global changes in gene expression patterns.

C-terminal deletions of the Escherichia coli RecA protein. Characterization of in vivo and in vitro effects.

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.

RecA Protein from the extremely radioresistant bacterium Deinococcus radiodurans: expression, purification, and characterization.

The RecA protein of Deinococcus radiodurans (RecA(Dr)) is essential for the extreme radiation resistance of this organism. The RecA(Dr) protein has been cloned and expressed in Escherichia coli and purified from this host. In some respects, the RecA(Dr) protein and the E. coli RecA (RecA(Ec)) proteins are close functional homologues. RecA(Dr) forms filaments on single-stranded DNA (ssDNA) that are similar to those formed by the RecA(Ec). The RecA(Dr) protein hydrolyzes ATP and dATP and promotes DNA strand exchange reactions. DNA strand exchange is greatly facilitated by the E. coli SSB protein. As is the case with the E. coli RecA protein, the use of dATP as a cofactor permits more facile displacement of bound SSB protein from ssDNA. However, there are important differences as well. The RecA(Dr) protein promotes ATP- and dATP-dependent reactions with distinctly different pH profiles. Although dATP is hydrolyzed at approximately the same rate at pHs 7.5 and 8.1, dATP supports an efficient DNA strand exchange only at pH 8.1. At both pHs, ATP supports efficient DNA strand exchange through heterologous insertions but dATP does not. Thus, dATP enhances the binding of RecA(Dr) protein to ssDNA and the displacement of ssDNA binding protein, but the hydrolysis of dATP is poorly coupled to DNA strand exchange. The RecA(Dr) protein thus may offer new insights into the role of ATP hydrolysis in the DNA strand exchange reactions promoted by the bacterial RecA proteins. In addition, the RecA(Dr) protein binds much better to duplex DNA than the RecA(Ec) protein, binding preferentially to double-stranded DNA (dsDNA) even when ssDNA is present in the solutions. This may be of significance in the pathways for dsDNA break repair in Deinococcus.