Pif1 family helicases promote mutation avoidance during DNA replication.

Pif1 family 5' → 3' DNA helicases are important for replication fork progression and genome stability. The budding yeast Saccharomyces cerevisiae encodes two Pif1 family helicases, Rrm3 and Pif1, both of which are multi-functional. Here we describe novel functions for Rrm3 in promoting mutation avoidance during DNA replication. We show that loss of RRM3 results in elevated spontaneous mutations made by DNA polymerases Pols ϵ and δ, which are subject to DNA mismatch repair. The absence of RRM3 also causes higher mutagenesis by the fourth B-family DNA polymerase Pol ζ. By genome-wide analysis, we show that the mutational consequences due to loss of RRM3 vary depending on the genomic locus. Rrm3 promotes the accuracy of DNA replication by Pols ϵ and δ across the genome, and it is particularly important for preventing Pol ζ-dependent mutagenesis at tRNA genes. In addition, mutation avoidance by Rrm3 depends on its helicase activity, and Pif1 serves as a backup for Rrm3 in suppressing mutagenesis. We present evidence that the sole human Pif1 family helicase in human cells likely also promotes replication fidelity, suggesting that a role for Pif1 family helicases in mutation avoidance may be evolutionarily conserved, a possible underlying mechanism for its potential tumor-suppressor function.

Rif1 Binding and Control of Chromosome-Internal DNA Replication Origins Is Limited by Telomere Sequestration.

The Saccharomyces cerevisiae telomere-binding protein Rif1 plays an evolutionarily conserved role in control of DNA replication timing by promoting PP1-dependent dephosphorylation of replication initiation factors. However, ScRif1 binding outside of telomeres has never been detected, and it has thus been unclear whether Rif1 acts directly on the replication origins that it controls. Here, we show that, in unperturbed yeast cells, Rif1 primarily regulates late-replicating origins within 100 kb of a telomere. Using the chromatin endogenous cleavage ChEC-seq technique, we robustly detect Rif1 at late-replicating origins that we show are targets of its inhibitory action. Interestingly, abrogation of Rif1 telomere association by mutation of its Rap1-binding module increases Rif1 binding and origin inhibition elsewhere in the genome. Our results indicate that Rif1 inhibits replication initiation by interacting directly with origins and suggest that Rap1-dependent sequestration of Rif1 increases its effective concentration near telomeres, while limiting its action at chromosome-internal sites.

Visualization of recombination-mediated damage bypass by template switching.

Template switching (TS) mediates damage bypass via a recombination-related mechanism involving PCNA polyubiquitination and polymerase δ-dependent DNA synthesis. Using two-dimensional gel electrophoresis and EM, here we characterize TS intermediates arising in Saccharomyces cerevisiae at a defined chromosome locus, identifying five major families of intermediates. Single-stranded DNA gaps of 150-200 nt, and not DNA ends, initiate TS by strand invasion. This causes reannealing of the parental strands and exposure of the nondamaged newly synthesized chromatid, which serves as a replication template for the other blocked nascent strand. Structures resembling double Holliday junctions, postulated to be central double-strand break-repair intermediates but so far visualized only in meiosis, mediate late stages of TS before being processed to hemicatenanes. Our results reveal the DNA transitions accounting for recombination-mediated DNA-damage tolerance in mitotic cells and replication under conditions of genotoxic stress.

BRCA1 controls homologous recombination at Tus/Ter-stalled mammalian replication forks.

Replication fork stalling can promote genomic instability, predisposing to cancer and other diseases. Stalled replication forks may be processed by sister chromatid recombination (SCR), generating error-free or error-prone homologous recombination (HR) outcomes. In mammalian cells, a long-standing hypothesis proposes that the major hereditary breast/ovarian cancer predisposition gene products, BRCA1 and BRCA2, control HR/SCR at stalled replication forks. Although BRCA1 and BRCA2 affect replication fork processing, direct evidence that BRCA gene products regulate homologous recombination at stalled chromosomal replication forks is lacking, due to a dearth of tools for studying this process. Here we report that the Escherichia coli Tus/Ter complex can be engineered to induce site-specific replication fork stalling and chromosomal HR/SCR in mouse cells. Tus/Ter-induced homologous recombination entails processing of bidirectionally arrested forks. We find that the Brca1 carboxy (C)-terminal tandem BRCT repeat and regions of Brca1 encoded by exon 11-two Brca1 elements implicated in tumour suppression-control Tus/Ter-induced homologous recombination. Inactivation of either Brca1 or Brca2 increases the absolute frequency of 'long-tract' gene conversions at Tus/Ter-stalled forks, an outcome not observed in response to a site-specific endonuclease-mediated chromosomal double-strand break. Therefore, homologous recombination at stalled forks is regulated differently from homologous recombination at double-strand breaks arising independently of a replication fork. We propose that aberrant long-tract homologous recombination at stalled replication forks contributes to genomic instability and breast/ovarian cancer predisposition in BRCA mutant cells.

Visualization and interpretation of eukaryotic DNA replication intermediates in vivo by electron microscopy.

The detailed understanding of the DNA replication process requires structural insight. The combination of psoralen cross-linking and electron microscopy has been extensively exploited to reveal the fine architecture of in vivo DNA replication intermediates. This approach proved instrumental to uncover the basic mechanisms of DNA duplication, as well as the perturbation of this process by various forms of replication stress. The replication structures are stabilized in vivo (by psoralen cross-linking) prior to extraction and enrichment procedures, allowing their visualization at the transmission electron microscope. This chapter outlines the procedures required to visualize and interpret in vivo replication intermediates of genomic DNA, extracted from budding yeast, Xenopus egg extracts, or cultured mammalian cells.

Combined bidimensional electrophoresis and electron microscopy to study specific plasmid DNA replication intermediates in human cells.

Replication interference by specific chromosomal sequences-such as trinucleotide repeats-plays a causative, though undefined role in the aetiology of human disease, especially neurodegenerative syndromes. However, studies on these mechanisms in human cells have been hampered by poorly defined replication origins on genomic DNA. Simian Virus 40 (SV40)-based plasmids were useful in the past to overcome these experimental limits, but have been rarely amenable for the most complex and revealing molecular biology approaches to study in vivo DNA replication interference. This chapter describes a new, safe, SV40-based episomal system that replicates with very high efficiency in human cells and allows isolation of in vivo replication intermediates with high yield and purity. We describe how to use this experimental system to run preparative agarose 2D-gel and to extract specific replication intermediates to visualize by electron microscopy.

Friedreich's ataxia-associated GAA repeats induce replication-fork reversal and unusual molecular junctions.

Expansion of GAA/TTC repeats is the causative event in Friedreich's ataxia. GAA repeats have been shown to hinder replication in model systems, but the mechanisms of replication interference and expansion in human cells remained elusive. To study in vivo replication structures at GAA repeats, we designed a new plasmid-based system that permits the analysis of human replication intermediates by two-dimensional gel electrophoresis and EM. We found that replication forks transiently pause and reverse at long GAA/TTC tracts in both orientations. Furthermore, we identified replication-associated intramolecular junctions, located between GAA/TTC repeats and other homopurine-homopyrimidine tracts, that were associated with breakage of the plasmid fork not traversing the repeats. Finally, we detected postreplicative, sister-chromatid hemicatenanes on control plasmids, which were replaced by persistent homology-driven junctions at GAA/TTC repeats. These data prove that GAA/TTC tracts interfere with replication in humans and implicate postreplicative mechanisms in trinucleotide repeat expansion.

Noncanonical mismatch repair as a source of genomic instability in human cells.

Mismatch repair (MMR) is a key antimutagenic process that increases the fidelity of DNA replication and recombination. Yet genetic experiments showed that MMR is required for antibody maturation, a process during which the immunoglobulin loci of antigen-stimulated B cells undergo extensive mutagenesis and rearrangements. In an attempt to elucidate the mechanism underlying the latter events, we set out to search for conditions that compromise MMR fidelity. Here, we describe noncanonical MMR (ncMMR), a process in which the MMR pathway is activated by various DNA lesions rather than by mispairs. ncMMR is largely independent of DNA replication, lacks strand directionality, triggers PCNA monoubiquitylation, and promotes recruitment of the error-prone polymerase-η to chromatin. Importantly, ncMMR is not limited to B cells but occurs also in other cell types. Moreover, it contributes to mutagenesis induced by alkylating agents. Activation of ncMMR may therefore play a role in genomic instability and cancer.

Exo1 competes with repair synthesis, converts NER intermediates to long ssDNA gaps, and promotes checkpoint activation.

Ultraviolet (UV) light induces DNA-damage checkpoints and mutagenesis, which are involved in cancer protection and tumorigenesis, respectively. How cells identify DNA lesions and convert them to checkpoint-activating structures is a major question. We show that during repair of UV lesions in noncycling cells, Exo1-mediated processing of nucleotide excision repair (NER) intermediates competes with repair DNA synthesis. Impediments of the refilling reaction allow Exo1 to generate extended ssDNA gaps, detectable by electron microscopy, which drive Mec1 kinase activation and will be refilled by long-patch repair synthesis, as shown by DNA combing. We provide evidence that this mechanism may be stimulated by closely opposing UV lesions, represents a strategy to redirect problematic repair intermediates to alternative repair pathways, and may also be extended to physically different DNA damages. Our work has significant implications for understanding the coordination between repair of DNA lesions and checkpoint pathways to preserve genome stability.