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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.

Understanding molecular mechanisms in the context of living cells requires the development of new methods of in vivo biochemical analysis to complement established in vitro biochemistry. A critically important molecular mechanism is genetic recombination, required for the beneficial reassortment of genetic information and for DNA double-strand break repair (DSBR). Central to recombination is the RecA (Rad51) protein that assembles into a spiral filament on DNA and mediates genetic exchange. Here we have developed a method that combines chromatin immunoprecipitation with next-generation sequencing (ChIP-Seq) and mathematical modeling to quantify RecA protein binding during the active repair of a single DSB in the chromosome of Escherichia coli. We have used quantitative genomic analysis to infer the key in vivo molecular parameters governing RecA loading by the helicase/nuclease RecBCD at recombination hot-spots, known as Chi. Our genomic analysis has also revealed that DSBR at the lacZ locus causes a second RecBCD-mediated DSBR event to occur in the terminus region of the chromosome, over 1 Mb away.

The repair of DNA double-strand breaks must be accurate to avoid genomic rearrangements that can lead to cell death and disease. This can be accomplished by promoting homologous recombination between correctly aligned sister chromosomes. Here, using a unique system for generating a site-specific DNA double-strand break in one copy of two replicating Escherichia coli sister chromosomes, we analyse the intermediates of sister-sister double-strand break repair. Using two-dimensional agarose gel electrophoresis, we show that when double-strand breaks are formed in the absence of RuvAB, 4-way DNA (Holliday) junctions are accumulated in a RecG-dependent manner, arguing against the long-standing view that the redundancy of RuvAB and RecG is in the resolution of Holliday junctions. Using pulsed-field gel electrophoresis, we explain the redundancy by showing that branch migration catalysed by RuvAB and RecG is required for stabilising the intermediates of repair as, when branch migration cannot take place, repair is aborted and DNA is lost at the break locus. We demonstrate that in the repair of correctly aligned sister chromosomes, an unstable early intermediate is stabilised by branch migration. This reliance on branch migration may have evolved to help promote recombination between correctly aligned sister chromosomes to prevent genomic rearrangements.

Approximately half the human genome is composed of repetitive DNA sequences classified into microsatellites, minisatellites, tandem repeats, and dispersed repeats. These repetitive sequences have coevolved within the genome but little is known about their potential interactions. Trinucleotide repeats (TNRs) are a subclass of microsatellites that are implicated in human disease. Expansion of CAG·CTG TNRs is responsible for Huntington disease, myotonic dystrophy, and a number of spinocerebellar ataxias. In yeast DNA double-strand break (DSB) formation has been proposed to be associated with instability and chromosome fragility at these sites and replication fork reversal (RFR) to be involved either in promoting or in preventing instability. However, the molecular basis for chromosome fragility of repetitive DNA remains poorly understood. Here we show that a CAG·CTG TNR array stimulates instability at a 275-bp tandem repeat located 6.3 kb away on the Escherichia coli chromosome. Remarkably, this stimulation is independent of both DNA double-strand break repair (DSBR) and RFR but is dependent on a functional mismatch repair (MMR) system. Our results provide a demonstration, in a simple model system, that MMR at one type of repetitive DNA has the potential to influence the stability of another. Furthermore, the mechanism of this stimulation places a limit on the universality of DSBR or RFR models of instability and chromosome fragility at CAG·CTG TNR sequences. Instead, our data suggest that explanations of chromosome fragility should encompass the possibility of chromosome gaps formed during MMR.

Structural maintenance of chromosomes (SMC) proteins have diverse cellular functions including chromosome segregation, condensation and DNA repair. They are grouped based on a conserved set of distinct structural motifs. All SMC proteins are predicted to have a bipartite ATPase domain that is separated by a long region predicted to form a coiled coil. Recent structural data on a variety of SMC proteins shows them to be arranged as long intramolecular coiled coils with a globular ATPase at one end. SMC proteins function in pairs as heterodimers or as homodimers often in complexes with other proteins. We expect the arrangement of the SMC protein domains in complex assemblies to have important implications for their diverse functions. We used scanning force microscopy imaging to determine the architecture of human, Saccharomyces cerevisiae, and Pyrococcus furiosus Rad50/Mre11, Escherichia coli SbcCD, and S.cerevisiae SMC1/SMC3 cohesin SMC complexes. Two distinct architectural arrangements are described, based on the way their components were connected. The eukaryotic complexes were similar to each other and differed from their prokaryotic and archaeal homologs. These similarities and differences are discussed with respect to their diverse mechanistic roles in chromosome metabolism.

A potentially lethal form of DNA/RNA modification, a cleavage complex, occurs when a nucleic acid-processing enzyme that acts via a transient covalent intermediate becomes trapped at its site of action. A number of overlapping pathways act to repair these lesions and many of the enzymes involved are those that catalyze recombinational-repair processes. A protein, Tdp1, has been identified that reverses cleavage-complex formation by specifically hydrolyzing a tyrosyl-DNA phosphodiester bond. The study of these pathways is both interesting and pertinent as they modulate the effectiveness of many antitumor/antibacterial drugs that act by stabilizing cleavage-complexes in vivo.

The recombination mechanisms that deal with double-strand breaks in organisms as diverse as phage, bacteria, yeast, and humans are remarkably conserved. We discuss conservation in the biochemical pathways required to recombine DNA ends and in the structure of the DNA products. In addition, we highlight that two fundamentally distinct broken DNA substrates exist and describe how they are repaired differently by recombination. Finally, we discuss the need to coordinate recombinational repair with cell division through DNA damage response pathways.

DNA double-strand break repair can be accomplished by homologous recombination when a sister chromatid or a homologous chromosome is available. However, the study of sister chromatid double-strand break repair in prokaryotes is complicated by the difficulty in targeting a break to only one copy of two essentially identical DNA sequences. We have developed a system using the Escherichia coli chromosome and the restriction enzyme EcoKI, in which double-strand breaks can be introduced into only one sister chromatid. We have shown that the components of the RecBCD and RecFOR 'pathways' are required for the recombinational repair of these breaks. Furthermore, we have shown a requirement for SbcCD, the prokaryotic homologue of Rad50/Mre11. This is the first demonstration that, like Rad50/Mre11, SbcCD is required for recombination in a wild-type cell. Our work suggests that the SbcCD-Rad50/Mre11 family of proteins, which have two globular domains separated by a long coiled-coil linker, is specifically required for the co-ordination of double-strand break repair reactions in which two DNA ends are required to recombine at one target site.

The Holliday junction is a central intermediate in homologous recombination. It consists of a four-way structure that can be resolved by cleavage to give either the crossover or noncrossover products observed. We show here that the formation of these products is controlled by the E. coli resolvasome (RuvABC) in such way that double-strand break repair (DSBR) leads to crossing over and single-strand gap repair (SSGR) does not lead to crossing over. We argue that the positioning of the RuvABC complex and its consequent direction of junction-cleavage is not random. In fact, the action of the RuvABC complex avoids crossing over in the most commonly predicted situations where Holliday junctions are encountered in DNA replication and repair. Our observations suggest that the positioning of the resolvasome may provide a general biochemical mechanism by which cells can control crossing over in recombination.

The expansion of normally polymorphic CTG microsatellites in certain human genes has been identified as the causative mutation of a number of hereditary neurological disorders, including Huntington's disease and myotonic dystrophy. Here, we have investigated the effect of methyl-directed mismatch repair (MMR) on the stability of a (CTG)43 repeat in Escherichia coli over 140 generations and find two opposing effects. In contrast to orientation-dependent repeat instability in wild-type E. coli and yeast, we observed no orientation dependence in MMR- E. coli cells and suggest that, for the repeat that we have studied, orientation dependence in wild-type cells is mainly caused by functional mismatch repair genes. Our results imply that slipped structures are generated during replication, causing single triplet expansions and contractions in MMR- cells, because they are left unrepaired. On the other hand, we find that the repair of such slipped structures by the MMR system can go awry, resulting in large contractions. We show that these mutS-dependent contractions arise preferentially when the CTG sequence is encoded by the lagging strand. The nature of this orientation dependence argues that the small slipped structures that are recognized by the MMR system are formed primarily on the lagging strand of the replication fork. It also suggests that, in the presence of functional MMR, removal of 3 bp slipped structures causes the formation of larger contractions that are probably the result of secondary structure formation by the CTG sequence. We rationalize the opposing effects of MMR on repeat tract stability with a model that accounts for CTG repeat instability and loss of orientation dependence in MMR- cells. Our work resolves a contradiction between opposing claims in the literature of both stabilizing and destabilizing effects of MMR on CTG repeat instability in E. coli.

We report here that homologous recombination functions are required for the viability of Escherichia coli cells maintaining a 240 bp chromosomal inverted repeat (palindromic) sequence. Wild-type cells can successfully replicate this palindrome but recA, recB or recC mutants carrying the palindrome are unviable. The dependence on homologous recombination for cell viability is overcome in sbcC mutants. Directly repeated copies of the DNA containing the palindrome are rapidly resolved to single copies in wild-type cells but not in sbcC mutants. Our results suggest that double-strand breaks introduced at the palindromic DNA sequence by the SbcCD nuclease are repaired by homologous recombination. The repair is conservative and the palindrome is retained in the repaired chromosome. We conclude that SbcCD can attack secondary structures but that repair conserves the DNA sequence with the potential to fold.