Building up and breaking down: mechanisms controlling recombination during replication

The complete and faithful duplication of the genome is an essential prerequisite for proliferating cells to maintain genome integrity. This objective is greatly challenged by DNA damage encountered during replication, which causes fork stalling and in certain cases, fork breakage. DNA damage tolerance (DDT) pathways mitigate the effects on fork stability induced by replication fork stalling by mediating damage-bypass and replication fork restart. These DDT mechanisms, largely relying on homologous recombination (HR) and specialized polymerases, can however contribute to genome rearrangements and mutagenesis. There is a profound connection between replication and recombination: recombination proteins protect replication forks from nuclease-mediated degradation of the nascent DNA strands and facilitate replication completion in cells challenged by DNA damage. Moreover, in case of fork collapse and formation of double strand breaks (DSBs), the recombination factors present or recruited to the fork facilitate HR-mediated DSB repair, which is primarily error-free. Disruption of HR is inexorably linked to genome instability, but the premature activation of HR during replication often leads to genome rearrangements. Faithful replication necessitates the downregulation of HR and disruption of active RAD51 filaments at replication forks, but upon persistent fork stalling, building up of HR is critical for the reorganization of the replication fork and for filling-in of the gaps associated with discontinuous replication induced by DNA lesions. Here we summarize and reflect on our understanding of the mechanisms that either suppress recombination or locally enhance it during replication, and the principles that underlie this regulation.     

Links between replication, recombination and genome instability in eukaryotes

Double-strand breaks in DNA can be repaired by homologous recombination including break-induced replication. In this reaction, the end of a broken DNA invades an intact chromosome and primes DNA replication resulting in the synthesis of an intact chromosome. Break-induced replication has also been suggested to cause different types of genome rearrangements.     

When replication forks stop

DNA synthesis is an accurate and very processive phenomenon, yet chromosome replication does not proceed at a constant rate and progression of the replication fork can be impeded. Several structural and functional features of the template can modulate the rate of progress of the replication fork. These include DNA secondary structures, DNA damage and occupied protein-binding sites. In addition, prokaryotes contain sites where replication is specifically arrested. DNA regions at which the replication machinery is blocked or transiently slowed could be particularly susceptible to genome rearrangements. Illegitimate recombination, a ubiquitous phenomenon which may have dramatic consequences, occurs by a variety of mechanisms. The observation that some rearrangements might be facilitated by a pause in replication could provide a clue in elucidating these processes. In support of this, some homologous and illegitimate recombination events have already been correlated with replication pauses or arrest sites.     

Elements of the polyomavirus replication origin required for homologous recombination mediated by large T antigen.

We introduced various elements of the polyomavirus origin of DNA replication into the genome of rat cells, and we analyzed their capacity to elicit rearrangements within the integrated sequences when exposed to large T antigen. The cis-acting sequences required for homologous recombination were those that make up a functional replication origin.     

Genome Rearrangements Caused by Depletion of Essential DNA Replication Proteins in Saccharomyces cerevisiae

Genetic screens of the collection of ～4500 deletion mutants in Saccharomyces cerevisiae have identified the cohort of nonessential genes that promote maintenance of genome integrity. Here we probe the role of essential genes needed for genome stability. To this end, we screened 217 tetracycline-regulated promoter alleles of essential genes and identified 47 genes whose depletion results in spontaneous DNA damage. We further showed that 92 of these 217 essential genes have a role in suppressing chromosome rearrangements. We identified a core set of 15 genes involved in DNA replication that are critical in preventing both spontaneous DNA damage and genome rearrangements. Mapping, classification, and analysis of rearrangement breakpoints indicated that yeast fragile sites, Ty retrotransposons, tRNA genes, early origins of replication, and replication termination sites are common features at breakpoints when essential replication genes that suppress chromosome rearrangements are downregulated. We propose mechanisms by which depletion of essential replication proteins can lead to double-stranded DNA breaks near these features, which are subsequently repaired by homologous recombination at repeated elements.     

Recovery of Arrested Replication Forks by Homologous Recombination Is Error-Prone

Homologous recombination is a universal mechanism that allows repair of DNA and provides support for DNA replication. Homologous recombination is therefore a major pathway that suppresses non-homology-mediated genome instability. Here, we report that recovery of impeded replication forks by homologous recombination is error-prone. Using a fork-arrest-based assay in fission yeast, we demonstrate that a single collapsed fork can cause mutations and large-scale genomic changes, including deletions and translocations. Fork-arrest-induced gross chromosomal rearrangements are mediated by inappropriate ectopic recombination events at the site of collapsed forks. Inverted repeats near the site of fork collapse stimulate large-scale genomic changes up to 1,500 times over spontaneous events. We also show that the high accuracy of DNA replication during S-phase is impaired by impediments to fork progression, since fork-arrest-induced mutation is due to erroneous DNA synthesis during recovery of replication forks. The mutations caused are small insertions/duplications between short tandem repeats (micro-homology) indicative of replication slippage. Our data establish that collapsed forks, but not stalled forks, recovered by homologous recombination are prone to replication slippage. The inaccuracy of DNA synthesis does not rely on PCNA ubiquitination or trans-lesion-synthesis DNA polymerases, and it is not counteracted by mismatch repair. We propose that deletions/insertions, mediated by micro-homology, leading to copy number variations during replication stress may arise by progression of error-prone replication forks restarted by homologous recombination.     

Epigenetic regulation of heterochromatic DNA stability

In this review we summarize recent studies that demonstrate the importance of epigenetic mechanisms for maintaining genome integrity, specifically with respect to repeated DNAs within heterochromatin. Potential problems that arise during replication, recombination, and repair of repeated sequences are counteracted by post-translational histone modifications and associated proteins, including the cohesins. These factors appear to ensure repeat stability by multiple mechanisms: suppressing homologous recombination, controlling the three-dimensional organization of damaged repeats to reduce the probability of aberrant recombination, and promoting the use of less problematic repair pathways. The presence of such systems may facilitate repeat and chromosome evolution, and their failure can lead to genome instability, chromosome rearrangements, and the onset of pathogenesis.     

Bacterial DNA repair genes and their eukaryotic homologues: 5. The role of recombination in DNA repair and genome stability

Recombinational repair is a well conserved DNA repair mechanism present in all living organisms. Repair by homologous recombination is generally accurate as it uses undamaged homologous DNA molecule as a repair template. In Escherichia coli homologous recombination repairs both the double-strand breaks and single-strand gaps in DNA. DNA double-strand breaks (DSB) can be induced upon exposure to exogenous sources such as ionizing radiation or endogenous DNA-damaging agents including reactive oxygen species (ROS) as well as during natural biological processes like conjugation. However, the bulk of double strand breaks are formed during replication fork collapse encountering an unrepaired single strand gap in DNA. Under such circumstances DNA replication on the damaged template can be resumed only if supported by homologous recombination. This functional cooperation of homologous recombination with replication machinery enables successful completion of genome duplication and faithful transmission of genetic material to a daughter cell. In eukaryotes, homologous recombination is also involved in essential biological processes such as preservation of genome integrity, DNA damage checkpoint activation, DNA damage repair, DNA replication, mating type switching, transposition, immune system development and meiosis. When unregulated, recombination can lead to genome instability and carcinogenesis.     

Suppression of Repeat-Mediated Gross Mitochondrial Genome Rearrangements by RecA in the Moss Physcomitrella patens

RecA and its ubiquitous homologs are crucial components in homologous recombination. Besides their eukaryotic nuclear counterparts, plants characteristically possess several bacterial-type RecA proteins localized to chloroplasts and/or mitochondria, but their roles are poorly understood. Here, we analyzed the role of the only mitochondrial RecA in the moss Physcomitrella patens. Disruption of the P. patens mitochondrial recA gene RECA1 caused serious defects in plant growth and development and abnormal mitochondrial morphology. Analyses of mitochondrial DNA in disruptants revealed that frequent DNA rearrangements occurred at multiple loci. Structural analysis suggests that the rearrangements, which in some cases were associated with partial deletions and amplifications of mitochondrial DNA, were due to aberrant recombination between short (<100 bp) direct and inverted repeats in which the sequences were not always identical. Such repeats are abundant in the mitochondrial genome, and interestingly many are located in group II introns. These results suggest that RECA1 does not promote but rather suppresses recombination among short repeats scattered throughout the mitochondrial genome, thereby maintaining mitochondrial genome stability. We propose that RecA-mediated homologous recombination plays a crucial role in suppression of short repeat-mediated genome rearrangements in plant mitochondria.     

Diversity of genome structure in Salmonella enterica serovar Typhi populations

The genomes of most strains of Salmonella and Escherichia coli are highly conserved. In contrast, all 136 wild-type strains of Salmonella enterica serovar Typhi analyzed by partial digestion with I-CeuI (an endonuclease which cuts within the rrn operons) and pulsed-field gel electrophoresis and by PCR have rearrangements due to homologous recombination between the rrn operons leading to inversions and translocations. Recombination between rrn operons in culture is known to be equally frequent in S. enterica serovar Typhi and S. enterica serovar Typhimurium; thus, the recombinants in S. enterica serovar Typhi, but not those in S. enterica serovar Typhimurium, are able to survive in nature. However, even in S. enterica serovar Typhi the need for genome balance and the need for gene dosage impose limits on rearrangements. Of 100 strains of genome types 1 to 6, 72 were only 25.5 kb off genome balance (the relative lengths of the replichores during bidirectional replication from oriC to the termination of replication [Ter]), while 28 strains were less balanced (41 kb off balance), indicating that the survival of the best-balanced strains was greater. In addition, the need for appropriate gene dosage apparently selected against rearrangements which moved genes from their accustomed distance from oriC. Although rearrangements involving the seven rrn operons are very common in S. enterica serovar Typhi, other duplicated regions, such as the 25 IS200 elements, are very rarely involved in rearrangements. Large deletions and insertions in the genome are uncommon, except for deletions of Salmonella pathogenicity island 7 (usually 134 kb) from fragment I-CeuI-G and 40-kb insertions, possibly a prophage, in fragment I-CeuI-E. The phage types were determined, and the origins of the phage types appeared to be independent of the origins of the genome types.     

Smc5-Smc6 mediate DNA double-strand-break repair by promoting sister-chromatid recombination

DNA double-strand breaks (DSB) can arise during DNA replication, or after exposure to DNA-damaging agents, and their correct repair is fundamental for cell survival and genomic stability. Here, we show that the Smc5-Smc6 complex is recruited to DSBs de novo to support their repair by homologous recombination between sister chromatids. In addition, we demonstrate that Smc5-Smc6 is necessary to suppress gross chromosomal rearrangements. Our findings show that the Smc5-Smc6 complex is essential for genome stability as it promotes repair of DSBs by error-free sister-chromatid recombination (SCR), thereby suppressing inappropriate non-sister recombination events.     

Schizosaccharomyces pombe Mms1 channels repair of perturbed replication into Rhp51 independent homologous recombination

In both Schizosaccharomyces pombe and Saccharomyces cerevisiae, Mms22 and Mms1 form a complex with important functions in the response to DNA damage, loss of which leads to perturbations during replication. Furthermore, in S. cerevisiae, Mms1 has been suggested to function in concert with a Cullin-like protein, Rtt101/Cul8, a potential paralog of Cullin 4. We performed epistasis analysis between Δmms1 and mutants of pathways with known functions in genome integrity, and measured the recruitment of homologous recombination proteins to blocked replication forks and recombination frequencies. We show that, in S. pombe, the functions of Mms1 and the conserved components of the Cullin 4 ubiquitin ligase, Pcu4 and Ddb1, do not significantly overlap. Furthermore, unlike in S. cerevisiae, the function of the H3K56 acetylase Rtt109 is not essential for Mms1 function. We provide evidence that Mms1 function is particularly important when a single strand break is converted into a double strand break during replication. Genetic data connect Mms1 to a Mus81 and Rad22(Rad52) dependent, but Rhp51 independent, branch of homologous recombination. This is supported by results demonstrating that Mms1 is recruited to a site-specific replication fork barrier and that, in a Δmms1 strain, Rad22(Rad52) and RPA recruitment to blocked forks are reduced, whereas Rhp51 recruitment is unaffected. In addition, Mms1 appears to specifically promote chromosomal rearrangements in a recombination assay. These observations suggest that Mms1 acts to channel repair of perturbed replication into a particular sub-pathway of homologous recombination.     

Tumor-specific gene expression in hepatic metastases by a replication-activated adenovirus vector

Clinical applications of tumor gene therapy require tumor-specific delivery or expression of therapeutic genes in order to maximize the oncolytic index and minimize side effects. This study demonstrates activation of transgene expression exclusively in hepatic metastases after systemic application of a modified first-generation (E1A/E1B-deleted) adenovirus vector (AdE1-) in mouse tumor models. The discrimination between tumors and normal liver tissue is based on selective DNA replication of AdE1- vectors in tumor cells. This new AdE1- based vector system uses homologous recombination between inverted repeats to mediate precise rearrangements within the viral genome. As a result of these rearrangements, a promoter is brought into conjunction with a reporter gene creating a functional expression cassette. Genomic rearrangements are dependent upon viral DNA replication, which in turn occurs specifically in tumor cells. In a mouse tumor model with liver metastases derived from human tumor cells, a single systemic administration of replication activated AdE1- vectors achieved transgene expression in every metastasis, whereas no extra-tumoral transgene induction was observed. Here we provide a new concept for tumor-specific gene expression that is also applicable for other conditionally replicating adenovirus vectors.     

Recombination and Replication

The links between recombination and replication have been appreciated for decades and it is now generally accepted that these two fundamental aspects of DNA metabolism are inseparable: Homologous recombination is essential for completion of DNA replication and vice versa. This review focuses on the roles that recombination enzymes play in underpinning genome duplication, aiding replication fork movement in the face of the many replisome barriers that challenge genome stability. These links have many conserved features across all domains of life, reflecting the conserved nature of the substrate for these reactions, DNA.The interplay between replication and recombination is complex in terms of both mechanism and integration within DNA metabolism. At the heart of this interplay is the requirement for single-stranded DNA (ssDNA), the substrate for DNA-strand-exchange proteins, to initiate recombination (Cox 2007b; San Filippo et al. 2008). Whether, when, and where this ssDNA is generated determines the functional relationship between replication and recombination, a relationship that can operate in both directions. Homologous recombination enzymes are critical for successful completion of genome duplication (Kogoma 1997; Cox et al. 2000) but DNA replication also underpins homologous recombination, as discussed elsewhere in this collection. The links between recombination and replication are therefore intimate and one cannot be considered in isolation from the other. However, involvement of DNA-strand-exchange proteins, regardless of the metabolic context, comes with the unavoidable risk of genome rearrangements. This genome instability can occasionally increase evolutionary fitness but more frequently is deleterious to the viability of the individual.This review will focus on fundamental aspects of the links between replication and recombination enzymes rather than simply providing a list of known enzymes and reactions. The substrate, DNA, is identical in all of these reactions and this is reflected in the high mechanistic conservation of replication and recombination.     

The Chromatin Assembly Factor 1 Promotes Rad51-Dependent Template Switches at Replication Forks by Counteracting D-Loop Disassembly by the RecQ-Type Helicase Rqh1

At blocked replication forks, homologous recombination mediates the nascent strands to switch template in order to ensure replication restart, but faulty template switches underlie genome rearrangements in cancer cells and genomic disorders. Recombination occurs within DNA packaged into chromatin that must first be relaxed and then restored when recombination is completed. The chromatin assembly factor 1, CAF-1, is a histone H3-H4 chaperone involved in DNA synthesis-coupled chromatin assembly during DNA replication and DNA repair. We reveal a novel chromatin factor-dependent step during replication-coupled DNA repair: Fission yeast CAF-1 promotes Rad51-dependent template switches at replication forks, independently of the postreplication repair pathway. We used a physical assay that allows the analysis of the individual steps of template switch, from the recruitment of recombination factors to the formation of joint molecules, combined with a quantitative measure of the resulting rearrangements. We reveal functional and physical interplays between CAF-1 and the RecQ-helicase Rqh1, the BLM homologue, mutations in which cause Bloom''s syndrome, a human disease associating genome instability with cancer predisposition. We establish that CAF-1 promotes template switch by counteracting D-loop disassembly by Rqh1. Consequently, the likelihood of faulty template switches is controlled by antagonistic activities of CAF-1 and Rqh1 in the stability of the D-loop. D-loop stabilization requires the ability of CAF-1 to interact with PCNA and is thus linked to the DNA synthesis step. We propose that CAF-1 plays a regulatory role during template switch by assembling chromatin on the D-loop and thereby impacting the resolution of the D-loop.     

Ensuring the fidelity of recombination in mammalian chromosomes

Mammalian cells frequently depend on homologous recombination (HR) to repair DNA damage accurately and to help rescue stalled or collapsed replication forks. The essence of HR is an exchange of nucleotides between identical or nearly identical sequences. Although HR fulfills important biological roles, recombination between inappropriate sequence partners can lead to translocations or other deleterious rearrangements and such events must be avoided. For example, the recombination machinery must follow stringent rules to preclude recombination between the many repetitive elements in a mammalian genome that share significant but imperfect homology. This paper takes a conceptual approach in addressing the homology requirements for recombination in mammalian genomes as well as the general strategy used by cells to reject recombination between similar but imperfectly matched sequences. A mechanism of heteroduplex rejection that involves the unwinding of recombination intermediates that may form between mismatched sequences is discussed.     

A Model Simulating the Dynamics of Plant Mitochondrial Genomes

Molecular evolution of the plant mitochondrial genome involves rearrangements due to the presence of highly recombining repeated sequences. As a result, this genome is composed of a set of molecules of various sizes that generate each other through recombination. The model presented simulates the evolution of various frequencies of the different types of molecules over successive cell cycles. It considers the mitochondrial genome as a population of circular molecules evolving through recombination, replication and random segregation. The model parameters are the rates of recombination of each sequence, the frequency of each type of recombination, the replication rates of the circles and the total amount of mitochondrial DNA per cell. This model demonstrates that high recombination rates lead to rapid deletions of sequences in the absence of selection. The frequency of deletion is dependent on the simulated reproductive mechanism. The conditions leading to reversible or irreversible rearrangements were also investigated.     

Srs2 mediates PCNA‐SUMO‐dependent inhibition of DNA repair synthesis

Completion of DNA replication needs to be ensured even when challenged with fork progression problems or DNA damage. PCNA and its modifications constitute a molecular switch to control distinct repair pathways. In yeast, SUMOylated PCNA (S‐PCNA) recruits Srs2 to sites of replication where Srs2 can disrupt Rad51 filaments and prevent homologous recombination (HR). We report here an unexpected additional mechanism by which S‐PCNA and Srs2 block the synthesis‐dependent extension of a recombination intermediate, thus limiting its potentially hazardous resolution in association with a cross‐over. This new Srs2 activity requires the SUMO interaction motif at its C‐terminus, but neither its translocase activity nor its interaction with Rad51. Srs2 binding to S‐PCNA dissociates Polδ and Polη from the repair synthesis machinery, thus revealing a novel regulatory mechanism controlling spontaneous genome rearrangements. Our results suggest that cycling cells use the Siz1‐dependent SUMOylation of PCNA to limit the extension of repair synthesis during template switch or HR and attenuate reciprocal DNA strand exchanges to maintain genome stability.     

High-frequency intermolecular homologous recombination during herpes simplex virus-mediated plasmid DNA replication

Homologous recombination is a prominent feature of herpes simplex virus (HSV) type 1 DNA replication. This has been demonstrated and traditionally studied in experimental settings where repeated sequences are present or are being introduced into a single molecule for subsequent genome isomerization. In the present study, we have designed a pair of unique HSV amplicon plasmids to examine in detail intermolecular homologous recombination (IM-HR) between these amplicon plasmids during HSV-mediated DNA replication. Our data show that IM-HR occurred at a very high frequency: up to 60% of the amplicon concatemers retrieved from virion particles underwent intermolecular homologous recombination. Such a high frequency of IM-HR required that both plasmids be replicated by HSV-mediated replication, as IM-HR events were not detected when either one or both plasmids were replicated by simian virus 40-mediated DNA replication, even with the presence of HSV infection. In addition, the majority of the homologous recombination events resulted in sequence replacement or targeted gene repair, while the minority resulted in sequence insertion. These findings imply that frequent intermolecular homologous recombination may contribute directly to HSV genome isomerization. In addition, HSV-mediated amplicon replication may be an attractive model for studying intermolecular homologous recombination mechanisms in general in a mammalian system. In this regard, the knowledge obtained from such a study may facilitate the development of better strategies for targeted gene correction for gene therapy purposes.