DNA damage tolerance plays a key role in protecting cell viability through translesion synthesis and template switching-mediated bypass of genotoxic polymerase-blocking base lesions. Both tolerance pathways critically rely on ubiquitylation of the proliferating-cell nuclear antigen (PCNA) on lysine 164 and have been proposed to operate uncoupled from replication. We report that Ubp10 and Ubp12 ubiquitin proteases differentially cooperate in PCNA deubiquitylation, owing to distinct activities on PCNA-linked ubiquitin chains. Ubp10 and Ubp12 associate with replication forks in a fashion determined by Ubp10 dependency on lagging-strand PCNA residence, and they downregulate translesion polymerase recruitment and template switch events engaging nascent strands. These findings reveal PCNAK164 deubiquitylation as a key mechanism for the modulation of lesion bypass during replication, which might set a framework for establishing strand-differential pathway choices. We propose that damage tolerance is tempered at replication forks to limit the extension of bypass events and sustain chromosome replication rates.

Transcription hinders replication fork progression and stability. The ATR checkpoint and specialized DNA helicases assist DNA synthesis across transcription units to protect genome integrity. Combining genomic and genetic approaches together with the analysis of replication intermediates, we searched for factors coordinating replication with transcription. We show that the Sen1/Senataxin DNA/RNA helicase associates with forks, promoting their progression across RNA polymerase II (RNAPII)-transcribed genes. sen1 mutants accumulate aberrant DNA structures and DNA-RNA hybrids while forks clash head-on with RNAPII transcription units. These replication defects correlate with hyperrecombination and checkpoint activation in sen1 mutants. The Sen1 function at the forks is separable from its role in RNA processing. Our data, besides unmasking a key role for Senataxin in coordinating replication with transcription, provide a framework for understanding the pathological mechanisms caused by Senataxin deficiencies and leading to the severe neurodegenerative diseases ataxia with oculomotor apraxia type 2 and amyotrophic lateral sclerosis 4.

DNA replication is sensitive to damage in the template. To bypass lesions and complete replication, cells activate recombination-mediated (error-free) and translesion synthesis-mediated (error-prone) DNA damage tolerance pathways. Crucial for error-free DNA damage tolerance is template switching, which depends on the formation and resolution of damage-bypass intermediates consisting of sister chromatid junctions. Here we show that a chromatin architectural pathway involving the high mobility group box protein Hmo1 channels replication-associated lesions into the error-free DNA damage tolerance pathway mediated by Rad5 and PCNA polyubiquitylation, while preventing mutagenic bypass and toxic recombination. In the process of template switching, Hmo1 also promotes sister chromatid junction formation predominantly during replication. Its C-terminal tail, implicated in chromatin bending, facilitates the formation of catenations/hemicatenations and mediates the roles of Hmo1 in DNA damage tolerance pathway choice and sister chromatid junction formation. Together, the results suggest that replication-associated topological changes involving the molecular DNA bender, Hmo1, set the stage for dedicated repair reactions that limit errors during replication and impact on genome stability.

Problems during DNA replication underlie genomic instability and drive malignant transformation. The DNA damage checkpoint stabilizes stalled replication forks thus counteracting aberrant fork transitions, DNA breaks and chromosomal rearrangements. We analyzed fork processing in checkpoint deficient cells by coupling psoralen crosslinking with replication intermediate two-dimensional gel analysis. This revealed a novel role for Exo1 nuclease in resecting reversed replication fork structures and counteracting the accumulation of aberrant intermediates resembling fork cleavage products. Genetic analyses demonstrated a functional interplay of Exo1 with Mus81, Dna2 and Sae2 nucleases in promoting cell survival following replication stress, suggestive of concerted nucleolytic processing of stalled forks. While Mus81 and other Structure Specific Endonucleases do not contribute to obvious collapsed fork transitions, Dna2 promotes reversed fork resection likely by facilitating Exo1 access to nascent strands. Instead, Sae2 cooperates with Exo1 in counteracting putative fork cleavage events linked to double strand breaks formation and increased gross chromosomal rearrangement rates. Our data indicate that in checkpoint deficient cells diverse nuclease activities interface to eliminate aberrant replication intermediates and prevent chromosome instability.

Transcription hinders replication fork progression and stability, and the Mec1/ATR checkpoint protects fork integrity. Examining checkpoint-dependent mechanisms controlling fork stability, we find that fork reversal and dormant origin firing due to checkpoint defects are rescued in checkpoint mutants lacking THO, TREX-2, or inner-basket nucleoporins. Gene gating tethers transcribed genes to the nuclear periphery and is counteracted by checkpoint kinases through phosphorylation of nucleoporins such as Mlp1. Checkpoint mutants fail to detach transcribed genes from nuclear pores, thus generating topological impediments for incoming forks. Releasing this topological complexity by introducing a double-strand break between a fork and a transcribed unit prevents fork collapse. Mlp1 mutants mimicking constitutive checkpoint-dependent phosphorylation also alleviate checkpoint defects. We propose that the checkpoint assists fork progression and stability at transcribed genes by phosphorylating key nucleoporins and counteracting gene gating, thus neutralizing the topological tension generated at nuclear pore gated genes.

Mec1ATR mediates the DNA damage response (DDR), integrating chromosomal signals and mechanical stimuli. We show that the PP2A phosphatases, ceramide-activated enzymes, couple cell metabolism with the DDR. Using genomic screens, metabolic analysis, and genetic and pharmacological studies, we found that PP2A attenuates the DDR and that three metabolic circuits influence the DDR by modulating PP2A activity. Irc21, a putative cytochrome b5 reductase that promotes the condensation reaction generating dihydroceramides (DHCs), and Ppm1, a PP2A methyltransferase, counteract the DDR by activating PP2A; conversely, the nutrient-sensing TORC1-Tap42 axis sustains DDR activation by inhibiting PP2A. Loss-of-function mutations in IRC21, PPM1, and PP2A and hyperactive tap42 alleles rescue mec1 mutants. Ceramides synergize with rapamycin, a TORC1 inhibitor, in counteracting the DDR. Hence, PP2A integrates nutrient-sensing and metabolic pathways to attenuate the Mec1ATR response. Our observations imply that metabolic changes affect genome integrity and may help with exploiting therapeutic options and repositioning known drugs.

THO/TREX is a conserved nuclear complex that functions in mRNP biogenesis and prevents transcription-associated recombination. Whether or not it has a ubiquitous role in the genome is unknown. Chromatin immunoprecipitation (ChIP)-chip studies reveal that the Hpr1 component of THO and the Sub2 RNA-dependent ATPase have genome-wide distributions at active ORFs in yeast. In contrast to RNA polymerase II, evenly distributed from promoter to termination regions, THO and Sub2 are absent at promoters and distributed in a gradual 5' → 3' gradient. This is accompanied by a genome-wide impact of THO-Sub2 deletions on expression of highly expressed, long and high G+C-content genes. Importantly, ChIP-chips reveal an over-recruitment of Rrm3 in active genes in THO mutants that is reduced by RNaseH1 overexpression. Our work establishes a genome-wide function for THO-Sub2 in transcription elongation and mRNP biogenesis that function to prevent the accumulation of transcription-mediated replication obstacles, including R-loops.

G-quadruplexes are four-stranded nucleic acid structures whose biological functions remain poorly understood. In the yeast S. cerevisiae, we report that G-quadruplexes form and, if not properly processed, pose a specific challenge to replication. We show that the G-quadruplex-prone CEB1 tandem array is tolerated when inserted near ARS305 replication origin in wild-type cells but is very frequently destabilized upon treatment with the potent Phen-DC(3) G-quadruplex ligand, or in the absence of the G-quadruplex-unwinding Pif1 helicase, only when the G-rich strand is the template of leading-strand replication. The orientation-dependent instability is associated with the formation of Rad51-Rad52-dependent X-shaped intermediates during replication detected by two-dimensional (2D) gels, and relies on the presence of intact G-quadruplex motifs in CEB1 and on the activity of ARS305. The asymmetrical behaviour of G-quadruplex prone sequences during replication has implications for their evolutionary dynamics within genomes, including the maintenance of G-rich telomeres.

DNA polymerase ε (Polε) carries out high-fidelity leading strand synthesis owing to its exonuclease activity. Polε polymerase and exonuclease activities are balanced, because of partitioning of nascent DNA strands between catalytic sites, so that net resection occurs when synthesis is impaired. In vivo, DNA synthesis stalling activates replication checkpoint kinases, which act to preserve the functional integrity of replication forks. We show that stalled Polε drives nascent strand resection causing fork functional collapse, averted via checkpoint-dependent phosphorylation. Polε catalytic subunit Pol2 is phosphorylated on serine 430, influencing partitioning between polymerase and exonuclease active sites. A phosphormimetic S430D change reduces exonucleolysis in vitro and counteracts fork collapse. Conversely, non-phosphorylatable pol2-S430A expression causes resection-driven stressed fork defects. Our findings reveal that checkpoint kinases switch Polε to an exonuclease-safe mode preventing nascent strand resection and stabilizing stalled replication forks. Elective partitioning suppression has implications for the diverse Polε roles in genome integrity maintenance.