SSB and the RecG DNA helicase: an intimate association to rescue a stalled replication fork

In E. coli, the regression of stalled DNA replication forks is catalyzed by the DNA helicase RecG. One means of gaining access to the fork is by binding to the single strand binding protein or SSB. This interaction occurs via the wedge domain of RecG and the intrinsically disordered linker (IDL) of SSB, in a manner similar to that of SH3 domains binding to PXXP motif‐containing ligands in eukaryotic cells. During loading, SSB remodels the wedge domain so that the helicase domains bind to the parental, duplex DNA, permitting the helicase to translocate using thermal energy. This translocation may be used to clear the fork of obstacles, prior to the initiation of fork regression.     

RecG Directs DNA Synthesis during Double-Strand Break Repair

Homologous recombination provides a mechanism of DNA double-strand break repair (DSBR) that requires an intact, homologous template for DNA synthesis. When DNA synthesis associated with DSBR is convergent, the broken DNA strands are replaced and repair is accurate. However, if divergent DNA synthesis is established, over-replication of flanking DNA may occur with deleterious consequences. The RecG protein of Escherichia coli is a helicase and translocase that can re-model 3-way and 4-way DNA structures such as replication forks and Holliday junctions. However, the primary role of RecG in live cells has remained elusive. Here we show that, in the absence of RecG, attempted DSBR is accompanied by divergent DNA replication at the site of an induced chromosomal DNA double-strand break. Furthermore, DNA double-stand ends are generated in a recG mutant at sites known to block replication forks. These double-strand ends, also trigger DSBR and the divergent DNA replication characteristic of this mutant, which can explain over-replication of the terminus region of the chromosome. The loss of DNA associated with unwinding joint molecules previously observed in the absence of RuvAB and RecG, is suppressed by a helicase deficient PriA mutation (priA300), arguing that the action of RecG ensures that PriA is bound correctly on D-loops to direct DNA replication rather than to unwind joint molecules. This has led us to put forward a revised model of homologous recombination in which the re-modelling of branched intermediates by RecG plays a fundamental role in directing DNA synthesis and thus maintaining genomic stability.     

Characterisation of the catalytically active form of RecG helicase

Replication of DNA is fraught with difficulty and chromosomes contain many lesions which may block movement of the replicative machinery. However, several mechanisms to overcome such problems are beginning to emerge from studies with Escherichia coli. An important enzyme in one or more of these mechanisms is the RecG helicase, which may target stalled replication forks to generate a four-stranded (Holliday) junction, thus facilitating repair and/or bypass of the original lesion. To begin to understand how RecG might catalyse regression of fork structures, we have analysed what the catalytically active form of the enzyme may be. We have found that RecG exists as a monomer in solution as measured by gel filtration but when bound to junction DNA the enzyme forms two distinct protein–DNA complexes that contain one and two protein molecules. However, mutant inhibition studies failed to provide any evidence that RecG acts as a multimer in vitro. Additionally, there was no evidence for cooperativity in the junction DNA-stimulated hydrolysis of ATP. These data suggest that RecG functions as a monomer to unwind junction DNA, which supports an ‘inchworm’ rather than an ‘active rolling’ mechanism of DNA unwinding. The observed in vivo inhibition of wild-type RecG by mutant forms of the enzyme was attributed to occlusion of the DNA target and correlates with the very low abundance of replication forks within an E.coli cell, even during rapid growth.     

Interactions between branched DNAs and peptide inhibitors of DNA repair

The RecG helicase of Escherichia coli unwinds both Holliday junction (HJ) and replication fork DNA substrates. Our lab previously identified and characterized peptides (WRWYCR and KWWCRW) that block the activity of RecG on these substrates. We determined that the peptides bind HJ DNA and prevent the binding of RecG. Herein, we present further evidence that the peptides are competitive inhibitors of RecG binding to its substrates. We have generated structural models of interactions between WRWYCR and a junction substrate. Using the fluorescent probe 2-aminopurine, we show that inhibitors interact with highest affinity with HJs (K_d = 14 nM) and ~4- to 9-fold more weakly with replication fork substrates. The fluorescence assay results agree with the structural model, and predict the molecular basis for interactions between HJ-trapping peptides and branched DNA molecules. Specifically, aromatic amino acids in the peptides stack with bases at the center of the DNA substrates. These interactions are stabilized by hydrogen bonds to the DNA and by intrapeptide interactions. These peptides inhibit several proteins involved in DNA repair in addition to RecG, have been useful as tools to dissect recombination, and possess antibiotic activity. Greater understanding of the peptides’ mechanism of action will further increase their utility.     

Are the SSB-Interacting Proteins RecO,RecG, PriA and the DnaB-Interacting Protein Rep Bound to Progressing Replication Forks in Escherichia coli?

In all organisms several enzymes that are needed upon replication impediment are targeted to replication forks by interaction with a replication protein. In most cases these proteins interact with the polymerase clamp or with single-stranded DNA binding proteins (SSB). In Escherichia coli an accessory replicative helicase was also shown to interact with the DnaB replicative helicase. Here we have used cytological observation of Venus fluorescent fusion proteins expressed from their endogenous loci in live E. coli cells to determine whether DNA repair and replication restart proteins that interact with a replication protein travel with replication forks. A custom-made microscope that detects active replisome molecules provided that they are present in at least three copies was used. Neither the recombination proteins RecO and RecG, nor the replication accessory helicase Rep are detected specifically in replicating cells in our assay, indicating that either they are not present at progressing replication forks or they are present in less than three copies. The Venus-PriA fusion protein formed foci even in the absence of replication forks, which prevented us from reaching a conclusion.     

Conservation of RecG activity from pathogens to hyperthermophiles

Maintaining the integrity of the genome is essential for the survival of all organisms. RecG helicase plays an important part in this process in Escherichia coli, promoting recombination and DNA repair, and providing ways to rescue stalled replication forks by way of a Holliday junction intermediate. We purified RecG proteins from three other species: two Gram-positive mesophiles, Bacillus subtilis and Streptococcus pneumoniae, and one extreme thermophile, Aquifex aeolicus. All three proteins bind and unwind replication fork and Holliday junction DNA molecules with efficiencies similar to the E. coli protein. Proteins from the Gram-positive species promote DNA repair in E. coli, indicating either that RecG acts alone or that any necessary protein-protein interactions are conserved. The S. pneumoniae RecG reduces plasmid copy number when expressed in E. coli, indicating that like the E. coli protein it unwinds plasmid R loop structures used to prime replication. This effect is not seen with B. subtilis RecG; the protein either lacks R loop unwinding activity or is compromised by having insufficient ATP. The A. aeolicus protein unwinds DNA well at 60 degrees C but is less efficient at 37 degrees C, explaining its inability to function in E. coli at this temperature. The N-terminal extension present in this protein was investigated and found to be dispensable for activity and thermo-stability. The results presented suggest that the role of RecG in DNA replication and repair is likely to be conserved throughout all bacteria, which underlines the importance of this protein in genome duplication and cell survival.     

Resolution of Joint Molecules by RuvABC and RecG Following Cleavage of the Escherichia coli Chromosome by EcoKI

DNA double-strand breaks can be repaired by homologous recombination involving the formation and resolution of Holliday junctions. In Escherichia coli, the RuvABC resolvasome and the RecG branch-migration enzyme have been proposed to act in alternative pathways for the resolution of Holliday junctions. Here, we have studied the requirements for RuvABC and RecG in DNA double-strand break repair after cleavage of the E. coli chromosome by the EcoKI restriction enzyme. We show an asymmetry in the ability of RuvABC and RecG to deal with joint molecules in vivo. We detect linear DNA products compatible with the cleavage-ligation of Holliday junctions by the RuvABC pathway but not by the RecG pathway. Nevertheless we show that the XerCD-mediated pathway of chromosome dimer resolution is required for survival regardless of whether the RuvABC or the RecG pathway is active, suggesting that crossing-over is a common outcome irrespective of the pathway utilised. This poses a problem. How can cells resolve joint molecules, such as Holliday junctions, to generate crossover products without cleavage-ligation? We suggest that the mechanism of bacterial DNA replication provides an answer to this question and that RecG can facilitate replication through Holliday junctions.     

Genetic analysis of Escherichia coli RadA: functional motifs and genetic interactions

The RadA/Sms protein is a RecA‐related protein found universally in eubacteria and plants, implicated in processing of recombination intermediates. Here we show that the putative Zn finger, Walker A motif, KNRXG motif and Lon protease homology domain of the Escherichia coli RadA protein are required for DNA damage survival. RadA is unlikely to possess protease activity as the putative active site serine is not required. Mutants in RadA have strong synergistic phenotypes with those in the branch migration protein RecG. Sensitivity of radA recG mutants to azidothymidine (AZT) can be rescued by blocking recombination with recA or recF mutations or by overexpression of RuvAB, suggesting that lethal recombination intermediates accumulate in the absence of RadA and RecG. Synthetic genetic interactions for survival to AZT or ciprofloxacin exposure were observed between RadA and known or putative helicases including DinG, Lhr, PriA, Rep, RuvAB, UvrD, YejH and YoaA. These represent the first affected phenotypes reported for Lhr, YejH and YoaA. The specificity of these effects sheds new light on the role of these proteins in DNA damage avoidance and repair and implicates a role in replication gap processing for DinG and YoaA and a role in double‐strand break repair for YejH.     

Stopped-Flow Measurement of Reaction Rate of Dihydroxyfumaric Acid with 2, 6-Dichlorophenolindophenol

The proline auxotrophic strain of Acetobacter aceti No. 1023 treated with CaCl₂ solution was transformed to the Pro⁺ phenotype at a frequency of up to 10²/μg DNA using chromosomal DNA prepared from the wild type prototrophic strain. The CaCl₂-treated cells of A. aceti No. 1023 could also be rendered competent for uptake of plasmid DNA. In an attempt to produce an appropriate cloning vector for A. aceti, the restriction patterns of the cryptic plasmids, pTA5001 A (23.5 Kb) and pTA5001B (23 Kb), found in A. aceti No. 1023 were determined. A selectable marker (ampicillin resistance) was introduced onto these cryptic plasmids by fusing them to vector pACYC177 from E. coli, using their single restriction site for XhoI. The hybrid plasmids generated could replicate in and confer ampicillin resistance to both A. aceti and E. coli. The maximum transformation frequency for A. aceti No. 1023 with these vectors was 10³/μg DNA.     

ATPase cycle and DNA unwinding kinetics of RecG helicase

The superfamily 2 bacterial helicase, RecG, is a monomeric enzyme with a role in DNA repair by reversing stalled replication forks. The helicase must act specifically and rapidly to prevent replication fork collapse. We have shown that RecG binds tightly and rapidly to four-strand oligonucleotide junctions, which mimic a stalled replication fork. The helicase unwinds such DNA junctions with a step-size of approximately four bases per ATP hydrolyzed. To gain an insight into this mechanism, we used fluorescent stopped-flow and quenched-flow to measure individual steps within the ATPase cycle of RecG, when bound to a DNA junction. The fluorescent ATP analogue, mantATP, was used throughout to determine the rate limiting steps, effects due to DNA and the main states in the cycle. Measurements, when possible, were also performed with unlabeled ATP to confirm the mechanism. The data show that the chemical step of hydrolysis is the rate limiting step in the cycle and that this step is greatly accelerated by bound DNA. The ADP release rate is similar to the cleavage rate, so that bound ATP and ADP would be the main states during the ATP cycle. Evidence is provided that the main structural rearrangements, which bring about DNA unwinding, are linked to these states.     

Is RecG a general guardian of the bacterial genome?

The RecG protein of Escherichia coli is a double-stranded DNA translocase that unwinds a variety of branched DNAs in vitro, including Holliday junctions, replication forks, D-loops and R-loops. Coupled with the reported pleiotropy of recG mutations, this broad range of potential targets has made it hard to pin down what the protein does in vivo, though roles in recombination and replication fork repair have been suggested. However, recent studies suggest that RecG provides a more general defence against pathological DNA replication. We have postulated that this is achieved through the ability of RecG to eliminate substrates that the replication restart protein, PriA, could otherwise exploit to re-replicate the chromosome. Without RecG, PriA triggers a cascade of events that interfere with the duplication and segregation of chromosomes. Here we review the studies that led us to this idea and to conclude that RecG may be both a specialist activity and a general guardian of the genome.     

Construction of New Shuttle Vectors for Acetbacter

Shuttle vector pMV301 was constructed by ligation of pMV102 found in A. aceti subsp. xylinum NBI 1002 to E. coli plasmid pACYC177. It is 6.0 kb in size, has unique restriction sites suitable for insertion of a foreign DNA fragment and confers ampicillin resistance to the Acetobacter host. This vector transforms A. aceti subsp. aceti 10-80S1 and industrial vinegar producer A. aceti subsp. xylinum NBI 1002 as well as E. coli. Various chimeric plasmids were also constructed by ligation of pMV102 to E. coli plasmids to examine the expression of drug resistance genes. In addition to the ampicillin resistance gene, resistance genes for kanamycin, chloramphenicol and tetracycline derived from E. coli plasmids were expressed in Acetobacter. Most of the constructed shuttle vectors were stably maintained in Acetobacter.     

Instability of inhibited replication forks in E. coli

Inhibiting the progress of replication forks in E. coli makes them susceptible to breakage. Broken replication forks are evidently reassembled by the RecBCD recombinational repair pathway. These findings explain a particular pattern of DNA degradation during inhibition of chromosomal replication, the role of recombination in the viability of mutants with displaced replication origin, and hyper-recombination observed in the Terminus of the E. coli chromosome in rnh mutants. Breakage and repair of inhibited replication forks could be the reason for the recombination-dependence of inducible stable DNA replication. A mechanism by which RecABCD-dependent recombination between very short inverted repeats may help E. coli to invert an operon, transcribed in the direction opposite to that of DNA replication, is discussed.     

Bacteriophage T4 UvsW protein is a helicase involved in recombination, repair and the regulation of DNA replication origins.

Bacteriophage T4 UvsW protein is involved in phage recombination, repair and the regulation of replication origins. Here, we provide evidence that UvsW functions as a helicase. First, expression of UvsW allows growth of an (otherwise inviable) Escherichia coli recG rnhA double mutant, consistent with UvsW being a functional analog of the RecG helicase. Second, UvsW contains helicase sequence motifs, and a substitution (K141R) in the Walker 'A' motif prevents growth of the E.coli recG rnhA double mutant. Third, UvsW, but not UvsW-K141R, inhibits replication from a T4 origin at which persistent RNA-DNA hybrids form and presumably trigger replication initiation. Fourth, mutations that inactivate UvsW and endonuclease VII (which cleaves DNA branches) synergistically block repair of double-strand breaks. These in vivo results are consistent with a model in which UvsW is a DNA helicase that catalyzes branch migration and dissociation of RNA-DNA hybrids. In support of this model, a partially purified GST/UvsW fusion protein, but not a GST/UvsW-K141R fusion, displays ssDNA-dependent ATPase activity and is able to unwind a branched DNA substrate.     

大肠杆菌细胞DNA复制、修复和重组途径的衔接

以大肠杆菌为例围绕相关领域的研究动态进行分析和总结.DNA复制、损伤修复和重组过程的相互作用关系研究是当今生命科学研究的前沿和热点之一.越来越多的研究表明,在分子水平上,DNA复制、损伤修复和重组过程既彼此独立,又相互依存.上述途径可以通过许多关键蛋白质之间的相互作用加以协调和整合,并籍此使遗传物质DNA得到有效的维护和忠实的传递.需要指出的是,基于许多细胞内关键蛋白及其功能在生物界中普遍保守性的事实,相信来自大肠杆菌有关DNA复制、修复和重组之间的研究成果也会对相关真核生物的研究提供借鉴.     

The intrinsically disordered linker of E. coli SSB is critical for the release from single‐stranded DNA

The Escherichia coli single stranded DNA binding protein (SSB) is crucial for DNA replication, recombination and repair. Within each process, it has two seemingly disparate roles: it stabilizes single‐stranded DNA (ssDNA) intermediates generated during DNA processing and, forms complexes with a group of proteins known as the SSB‐interactome. Key to both roles is the C‐terminal, one‐third of the protein, in particular the intrinsically disordered linker (IDL). Previously, they have shown using a series of linker deletion mutants that the IDL links both ssDNA and target protein binding by mediating interactions with the oligosaccharide/oligonucleotide binding fold in the target. In this study, they examine the role of the linker region in SSB function in a variety of DNA metabolic processes in vitro. Using the same linker mutants, the results show that in addition to association reactions (either DNA or protein), the IDL is critical for the release of SSB from DNA. This release can be under conditions of ssDNA competition or active displacement by a DNA helicase or recombinase. Consistent with their previous work these results indicate that SSB linker mutants are defective for SSB–SSB interactions, and when the IDL is removed a terminal SSB–DNA complex results. Formation of this complex inhibits downstream processing of DNA by helicases such as RecG or PriA as well as recombination, mediated by RecA. A model, based on the evidence herein, is presented to explain how the IDL acts in SSB function.     

PriA helicase and SSB interact physically and functionally

PriA helicase is the major DNA replication restart initiator in Escherichia coli and acts to reload the replicative helicase DnaB back onto the chromosome at repaired replication forks and D-loops formed by recombination. We have discovered that PriA-catalysed unwinding of branched DNA substrates is stimulated specifically by contact with the single-strand DNA binding protein of E.coli, SSB. This stimulation requires binding of SSB to the initial DNA substrate and is effected via a physical interaction between PriA and the C-terminus of SSB. Stimulation of PriA by the SSB C-terminus may act to ensure that efficient PriA-catalysed reloading of DnaB occurs only onto the lagging strand template of repaired forks and D-loops. Correlation between the DNA repair and recombination defects of strains harbouring an SSB C-terminal mutation with inhibition of this SSB–PriA interaction in vitro suggests that SSB plays a critical role in facilitating PriA-directed replication restart. Taken together with previous data, these findings indicate that protein–protein interactions involving SSB may coordinate replication fork reloading from start to finish.