SummaryPoly(ADP-ribose) is synthesized by PARP enzymes during the repair of stochastic DNA breaks. Surprisingly, however, we show that most if not all endogenous poly(ADP-ribose) is detected in normal S phase cells at sites of DNA replication. This S phase poly(ADP-ribose) does not result from damaged or misincorporated nucleotides or from DNA replication stress. Rather, perturbation of the DNA replication proteins LIG1 or FEN1 increases S phase poly(ADP-ribose) more than 10-fold, implicating unligated Okazaki fragments as the source of S phase PARP activity. Indeed, S phase PARP activity is ablated by suppressing Okazaki fragment formation with emetine, a DNA replication inhibitor that selectively inhibits lagging strand synthesis. Importantly, PARP activation during DNA replication recruits the single-strand break repair protein XRCC1, and human cells lacking PARP activity and/or XRCC1 are hypersensitive to FEN1 perturbation. Collectively, our data indicate that PARP1 is a sensor of unligated Okazaki fragments during DNA replication and facilitates their repair.

Summary Mammalian DNA base excision repair (BER) is accelerated by poly(ADP-ribose) polymerases (PARPs) and the scaffold protein XRCC1. PARPs are sensors that detect single-strand break intermediates, but the critical role of XRCC1 during BER is unknown. Here, we show that protein complexes containing DNA polymerase β and DNA ligase III that are assembled by XRCC1 prevent excessive engagement and activity of PARP1 during BER. As a result, PARP1 becomes “trapped” on BER intermediates in XRCC1-deficient cells in a manner similar to that induced by PARP inhibitors, including in patient fibroblasts from XRCC1-mutated disease. This excessive PARP1 engagement and trapping renders BER intermediates inaccessible to enzymes such as DNA polymerase β and impedes their repair. Consequently, PARP1 deletion rescues BER and resistance to base damage in XRCC1 −/− cells. These data reveal excessive PARP1 engagement during BER as a threat to genome integrity and identify XRCC1 as an “anti-trapper” that prevents toxic PARP1 activity.

Highlights d XRCC1 prevents endogenous PARP1 trapping during DNA base excision repair d PARP1 trapping impedes base excision repair and increases sensitivity to base damage d In the absence of PARP1, XRCC1 is dispensable for DNA base excision repair

Background: How faulty Okazaki fragments are repaired remains unclear. Results: The DNA annealing activity of Rad52 and sister chromatid cohesion are important in the repair of faulty Okazaki fragments. Conclusion: Rad52/Rad59-mediated sister chromatid recombination is a major means of repairing faulty Okazaki fragments. Significance: The faithful repair of faulty Okazaki fragments is critical for genome stability.

Genetic defects in the repair of DNA single-strand breaks (SSBs) can result in neurological disease triggered by toxic activity of the single-strand-break sensor protein PARP1. However, the mechanism(s) by which this toxic PARP1 activity triggers cellular dysfunction are unclear. Here we show that human cells lacking XRCC1 fail to rapidly recover transcription following DNA base damage, a phenotype also observed in patient-derived fibroblasts with XRCC1 mutations and Xrcc1−/− mouse neurons. This defect is caused by excessive/aberrant PARP1 activity during DNA base excision repair, resulting from the loss of PARP1 regulation by XRCC1. We show that aberrant PARP1 activity suppresses transcriptional recovery during base excision repair by promoting excessive recruitment and activity of the ubiquitin protease USP3, which as a result reduces the level of monoubiquitinated histones important for normal transcriptional regulation. Importantly, inhibition and/or deletion of PARP1 or USP3 restores transcriptional recovery in XRCC1−/− cells, highlighting PARP1 and USP3 as possible therapeutic targets in neurological disease.

Lagging strand synthesis is mechanistically far more complicated than leading strand synthesis because it involves multistep processes and requires considerably more enzymes and protein factors. Due to this complexity, multiple fail-safe factors are required to ensure successful replication of the lagging strand DNA. We attempted to identify novel factors that are required in the absence of the helicase activity of Dna2, an essential enzyme in Okazaki-fragment maturation. In this article, we identified Rim11, a GSK-3β-kinase homolog, as a multicopy suppressor of helicase-dead mutant (). Subsequent epistasis analysis revealed that Ume6 (a DNA binding protein, a downstream substrate of Rim11) also acted as a multicopy suppressor of the allele. We found that the interaction of Ume6 with the conserved histone deacetylase complex Sin3-Rpd3 and the catalytic activity of Rpd3 were indispensable for the observed suppression of the mutant. Moreover, multicopy suppression by Rim11/Ume6 requires the presence of sister-chromatid recombination mediated by Rad52/Rad59 proteins, but not vice versa. Interestingly, the overexpression of Rim11 or Ume6 also suppressed the MMS sensitivity of Δ. We also showed that the lethality of helicase-dead mutant was attributed to checkpoint activation and that decreased levels of deoxynucleotide triphosphates (dNTPs) by overexpressing Sml1 (an inhibitor of ribonucleotide reductase) rescued the mutant. We also present evidence that indicates Rim11/Ume6 works independently but in parallel with that of checkpoint inhibition, dNTP regulation, and sister-chromatid recombination. In conclusion, our results establish Rim11, Ume6, the histone deacetylase complex Sin3-Rpd3 and Sml1 as new factors important in the events of faulty lagging strand synthesis.

Dna2 can efficiently process 5 0 flaps containing DNA secondary structure using coordinated action of the three biochemical activities: the N-terminally encoded DNA-binding activity and the C-terminally encoded endonuclease and helicase activities. In this study, we investigated the cross talk among the three functional domains using a variety of dna2 mutant alleles and enzymes derived thereof. We found that disruption of the catalytic activities of Dna2 activated Dna2-dependent checkpoint, residing in the Nterminal domain. This checkpoint activity contributed to growth defects of dna2 catalytic mutants, revealing the presence of an intramolecular functional cross talk in Dna2. The N-terminal domain of Dna2 bound specifically to substrates that mimic DNA replication fork intermediates, including Holliday junctions. Using site-directed mutagenesis of the N-terminal domain of Dna2, we discovered that five consecutive basic amino acid residues were essential for the ability of Dna2 to bind hairpin DNA in vitro. Mutant cells expressing the dna2 allele containing all five basic residues substituted with alanine displayed three distinct phenotypes: (i) temperature-sensitive growth defects, (ii) bypass of S-phase arrest, and (iii) increased sensitivity to DNA-damaging agents. Taken together, our results indicate that the interplay between the N-terminal regulatory and C-terminal catalytic domains of Dna2 plays an important role in vivo, especially when cells are placed under replication stress.