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Poly (ADP-ribose) polymerase (PARP) 1 and 2 enzymatic inhibitors (PARPi) are promising cancer treatments. But recently, their use has been hindered by unexplained severe anemia and treatment-related leukemia. In addition to enzymatic inhibition, PARPi also trap PARP1 and 2 at DNA lesions. Here we report that, unlike Parp2 -/- mice, which develop normally, mice expressing catalytically inactive Parp2 (E534A and Parp2 EA/EA ) succumb to Tp53- and Chk2-dependent erythropoietic failure in utero, mirroring Lig1 -/- mice. While DNA damage mainly activates PARP1, we demonstrate that DNA replication activates PARP2 robustly. PARP2 is selectively recruited and activated by 5'-phosphorylated nicks (5'p-nicks), including those between Okazaki fragments, resolved by ligase 1 (Lig1) and Lig3. Inactive PARP2, but not its active form or absence, impedes Lig1- and Lig3-mediated ligation, causing dose-dependent replication fork collapse, which is detrimental to erythroblasts with ultra-fast forks. This PARylation-dependent structural function of PARP2 at 5'p-nicks explains the detrimental effects of PARP2 inactivation on erythropoiesis, shedding light on PARPi-induced anemia and the selection for TP53/CHK2 loss., Competing Interests: Declaration of interests D.M. is a scientific editor for Molecular Cell and therefore was not involved in the peer review or the decision-making process of this manuscript., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Finalization of eukaryotic nuclear DNA replication relies on DNA ligase 1 (LIG1) to seal DNA nicks generated during Okazaki Fragment Maturation (OFM). Using a mutational reporter in Saccharomyces cerevisiae, we previously showed that mutation of the high-fidelity magnesium binding site of LIG1 Cdc9 strongly increases the rate of single-base insertions. Here we show that this rate is increased across the nuclear genome, that it is synergistically increased by concomitant loss of DNA mismatch repair (MMR), and that the additions occur in highly specific sequence contexts. These discoveries are all consistent with incorporation of an extra base into the nascent lagging DNA strand that can be corrected by MMR following mutagenic ligation by the Cdc9-EEAA variant. There is a strong preference for insertion of either dGTP or dTTP into 3-5 base pair mononucleotide sequences with stringent flanking nucleotide requirements. The results reveal unique LIG1 Cdc9 -dependent mutational motifs where high fidelity DNA ligation of a subset of OFs is critical for preventing mutagenesis across the genome., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Replication forks stalled at co-transcriptional R-loops can be restarted by a mechanism involving fork cleavage-religation cycles mediated by MUS81 endonuclease and DNA ligase IV (LIG4), which presumably relieve the topological barrier generated by the transcription-replication conflict (TRC) and facilitate ELL-dependent reactivation of transcription. Here, we report that the restart of R-loop-stalled replication forks via the MUS81-LIG4-ELL pathway requires senataxin (SETX), a helicase that can unwind RNA:DNA hybrids. We found that SETX promotes replication fork progression by preventing R-loop accumulation during S-phase. Interestingly, loss of SETX helicase activity leads to nascent DNA degradation upon induction of R-loop-mediated fork stalling by hydroxyurea. This fork degradation phenotype is independent of replication fork reversal and results from DNA2-mediated resection of MUS81-cleaved replication forks that accumulate due to defective replication restart. Finally, we demonstrate that SETX acts in a common pathway with the DEAD-box helicase DDX17 to suppress R-loop-mediated replication stress in human cells. A possible cooperation between these RNA/DNA helicases in R-loop unwinding at TRC sites is discussed., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

While DNA ligase I (LigI) joins most Okazaki fragments, a backup pathway involving poly(ADP-ribose) synthesis, XRCC1 and DNA ligase IIIα (LigIIIα) functions along with the LigI-dependent pathway and is also capable of supporting DNA replication in the absence of LigI. Here we have addressed for the first time the roles of PARP1 and PARP2 in this pathway using isogenic null derivatives of mouse CH12F3 cells. While single and double null mutants of the parental cell line and single mutants of LIG1 null cells were viable, loss of both PARP1 and PARP2 was synthetically lethal with LigI deficiency. Thus, PARP1 and PARP2 have a redundant essential role in LigI-deficient cells. Interestingly, higher levels of PARP2 but not PARP1 associated with newly synthesized DNA in the LIG1 null cells and there was a much higher increase in PARP2 chromatin retention in LIG1 null cells incubated with the PARP inhibitor olaparib with this effect occurring independently of PARP1. Together our results suggest that PARP2 plays a major role in specific cell types that are more dependent upon the backup pathway to complete DNA replication and that PARP2 retention at unligated Okazaki fragments likely contributes to the side effects of current clinical PARP inhibitors., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Synthesis and maturation of Okazaki Fragments is an incessant and highly efficient metabolic process completing the synthesis of the lagging strands at replication forks during S phase. Accurate Okazaki fragment maturation (OFM) is crucial to maintain genome integrity and, therefore, cell survival in all living organisms. In eukaryotes, OFM involves the consecutive action of DNA polymerase Pol ∂, 5' Flap endonuclease Fen1 and DNA ligase I, and constitutes the best example of a sequential process coordinated by the sliding clamp PCNA. For OFM to occur efficiently, cooperation of these enzymes with PCNA must be highly regulated. Here, we present evidence of a role for the K164-PCNA-deubiquitylase Ubp10 in the maturation of Okazaki fragments in the budding yeast Saccharomyces cerevisiae. We show that Ubp10 associates with lagging-strand DNA synthesis machineries on replicating chromatin to ensure timely ligation of Okazaki fragments by promoting PCNA dissociation from chromatin requiring lysine 164 deubiquitylation., (© 2024. The Author(s).)

Cyclosporin A (CsA) induces DNA double-strand breaks in LIG4 syndrome fibroblasts, specifically upon transit through S-phase. The basis underlying this has not been described. CsA-induced genomic instability may reflect a direct role of Cyclophilin A (CYPA) in DNA repair. CYPA is a peptidyl-prolyl cis-trans isomerase (PPI). CsA inhibits the PPI activity of CYPA. Using an integrated approach involving CRISPR/Cas9-engineering, siRNA, BioID, co-immunoprecipitation, pathway-specific DNA repair investigations as well as protein expression interaction analysis, we describe novel impacts of CYPA loss and inhibition on DNA repair. We characterise a direct CYPA interaction with the NBS1 component of the MRE11-RAD50-NBS1 complex, providing evidence that CYPA influences DNA repair at the level of DNA end resection. We define a set of genetic vulnerabilities associated with CYPA loss and inhibition, identifying DNA replication fork protection as an important determinant of viability. We explore examples of how CYPA inhibition may be exploited to selectively kill cancers sharing characteristic genomic instability profiles, including MYCN-driven Neuroblastoma, Multiple Myeloma and Chronic Myelogenous Leukaemia. These findings propose a repurposing strategy for Cyclophilin inhibitors., (© 2024. The Author(s).)

TIMELESS (TIM) in the fork protection complex acts as a scaffold of the replisome to prevent its uncoupling and ensure efficient DNA replication fork progression. Nevertheless, its underlying basis for coordinating leading and lagging strand synthesis to limit single-stranded DNA (ssDNA) exposure remains elusive. Here, we demonstrate that acute degradation of TIM at ongoing DNA replication forks induces the accumulation of ssDNA gaps stemming from defective Okazaki fragment (OF) processing. Cells devoid of TIM fail to support the poly(ADP-ribosyl)ation necessary for backing up the canonical OF processing mechanism mediated by LIG1 and FEN1. Consequently, recruitment of XRCC1, a known effector of PARP1-dependent single-strand break repair, to post-replicative ssDNA gaps behind replication forks is impaired. Physical disruption of the TIM-PARP1 complex phenocopies the rapid loss of TIM, indicating that the TIM-PARP1 interaction is critical for the activation of this compensatory pathway. Accordingly, combined deficiency of FEN1 and the TIM-PARP1 interaction leads to synergistic DNA damage and cytotoxicity. We propose that TIM is essential for the engagement of PARP1 to the replisome to coordinate lagging strand synthesis with replication fork progression. Our study identifies TIM as a synthetic lethal target of OF processing enzymes that can be exploited for cancer therapy., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

DNA ligase I (LigI), the predominant enzyme that joins Okazaki fragments, interacts with PCNA and Pol δ. LigI also interacts with UHRF1, linking Okazaki fragment joining with DNA maintenance methylation. Okazaki fragments can also be joined by a relatively poorly characterized DNA ligase IIIα (LigIIIα)-dependent backup pathway. Here we examined the effect of LigI-deficiency on proteins at the replication fork. Notably, LigI-deficiency did not alter the kinetics of association of the PCNA clamp, the leading strand polymerase Pol ε, DNA maintenance methylation proteins and core histones with newly synthesized DNA. While the absence of major changes in replication and methylation proteins is consistent with the similar proliferation rate and DNA methylation levels of the LIG1 null cells compared with the parental cells, the increased levels of LigIIIα/XRCC1 and Pol δ at the replication fork and in bulk chromatin indicate that there are subtle replication defects in the absence of LigI. Interestingly, the non-replicative histone H1 variant, H1.0, is enriched in the chromatin of LigI-deficient mouse CH12F3 and human 46BR.1G1 cells. This alteration was not corrected by expression of wild type LigI, suggesting that it is a relatively stable epigenetic change that may contribute to the immunodeficiencies linked with inherited LigI-deficiency syndrome., (© 2023. The Author(s).)

During lagging strand synthesis, DNA Ligase 1 (Lig1) cooperates with the sliding clamp PCNA to seal the nicks between Okazaki fragments generated by Pol δ and Flap endonuclease 1 (FEN1). We present several cryo-EM structures combined with functional assays, showing that human Lig1 recruits PCNA to nicked DNA using two PCNA-interacting motifs (PIPs) located at its disordered N-terminus (PIP N-term ) and DNA binding domain (PIP DBD ). Once Lig1 and PCNA assemble as two-stack rings encircling DNA, PIP N-term is released from PCNA and only PIP DBD is required for ligation to facilitate the substrate handoff from FEN1. Consistently, we observed that PCNA forms a defined complex with FEN1 and nicked DNA, and it recruits Lig1 to an unoccupied monomer creating a toolbelt that drives the transfer of DNA to Lig1. Collectively, our results provide a structural model on how PCNA regulates FEN1 and Lig1 during Okazaki fragments maturation., (© 2022. The Author(s).)

The recombination of short homologous ends in Escherichia coli has been known for 30 years, and it is often used for both site-directed mutagenesis and in vivo cloning. For cloning, a plasmid and target DNA fragments were converted into linear DNA fragments with short homologous ends, which are joined via recombination inside E. coli after transformation. Here this mechanism of joining homologous ends in E. coli was determined by a linearized plasmid with short homologous ends. Two 3'-5' exonucleases ExoIII and ExoX with nonprocessive activity digested linear dsDNA to generate 5' single-strand overhangs, which annealed with each other. The polymerase activity of DNA polymerase I (Pol I) was exclusively employed to fill in the gaps. The strand displacement activity and the 5'-3' exonuclease activity of Pol I were also required, likely to generate 5' phosphate termini for subsequent ligation. Ligase A (LigA) joined the nicks to finish the process. The model involving 5' single-stranded overhangs is different from established recombination pathways that all generate 3' single-stranded overhangs. This recombination is likely common in bacteria since the involved enzymes are ubiquitous., (© 2021 John Wiley & Sons Ltd.)

DNA ligase 1 (LIG1, Cdc9 in yeast) finalizes eukaryotic nuclear DNA replication by sealing Okazaki fragments using DNA end-joining reactions that strongly discriminate against incorrectly paired DNA substrates. Whether intrinsic ligation fidelity contributes to the accuracy of replication of the nuclear genome is unknown. Here, we show that an engineered low-fidelity LIG1 Cdc9 variant confers a novel mutator phenotype in yeast typified by the accumulation of single base insertion mutations in homonucleotide runs. The rate at which these additions are generated increases upon concomitant inactivation of DNA mismatch repair, or by inactivation of the Fen1 Rad27 Okazaki fragment maturation (OFM) nuclease. Biochemical and structural data establish that LIG1 Cdc9 normally avoids erroneous ligation of DNA polymerase slippage products, and this protection is compromised by mutation of a LIG1 Cdc9 high-fidelity metal binding site. Collectively, our data indicate that high-fidelity DNA ligation is required to prevent insertion mutations, and that this may be particularly critical following strand displacement synthesis during the completion of OFM.

To ensure genome integrity, the joining of breaks in the phosphodiester backbone of duplex DNA is required during DNA replication and to complete the repair of almost all types of DNA damage. In human cells, this task is accomplished by DNA ligases encoded by three genes, LIG1, LIG3 and LIG4. Mutations in LIG1 and LIG4 have been identified as the causative factor in two inherited immunodeficiency syndromes. Moreover, there is emerging evidence that DNA ligases may be good targets for the development of novel anti-cancer agents. In this graphical review, we provide an overview of the roles of the DNA ligases encoded by the three human LIG genes in DNA replication and repair., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Chronic hepatitis B virus (HBV) infections result in 887,000 deaths annually. The central challenge in curing HBV is eradication of the stable covalently closed circular DNA (cccDNA) form of the viral genome, which is formed by the repair of lesion-bearing HBV relaxed circular DNA delivered by the virions to hepatocytes. The complete and minimal set of host factors involved in cccDNA formation is unknown, largely due to the lack of a biochemical system that fully reconstitutes cccDNA formation. Here, we have developed experimental systems where various HBV relaxed-circular-DNA substrates are repaired to form cccDNA by both cell extracts and purified human proteins. Using yeast- and human-extract screenings, we identified five core components of lagging-strand synthesis as essential for cccDNA formation: proliferating cell nuclear antigen, the replication factor C complex, DNA polymerase δ, flap endonuclease 1 and DNA ligase 1. We reconstituted cccDNA formation with purified human homologues, establishing these as a minimal set of factors for cccDNA formation. We further demonstrated that treatment with the DNA-polymerase inhibitor aphidicolin diminishes cccDNA formation both in biochemical assays and in HBV-infected human cells. Together, our findings define key components in HBV cccDNA formation.

It is established that short inverted repeats trigger base substitution mutagenesis in human cells. However, how the replication machinery deals with structured DNA is unknown. It has been previously reported that in human cell-free extracts, DNA primer extension using a structured single-stranded template is transiently blocked at DNA hairpins. Here, the proteomic analysis of proteins bound to the DNA template is reported and evidence that the DNA-PK complex (DNA-PKcs and the Ku heterodimer) recognizes, and is activated by, structured single-stranded DNA is provided. Hijacking the DNA-PK complex by double-stranded oligonucleotides results in a large removal of the pausing sites and an elevated DNA extension efficiency. Conversely, DNA-PKcs inhibition results in its stabilization on the template, along with other proteins acting downstream in the Non-Homologous End-Joining (NHEJ) pathway, especially the XRCC4-DNA ligase 4 complex and the cofactor PAXX. Retention of NHEJ factors to the DNA in the absence of DNA-PKcs activity correlates with additional halts of primer extension, suggesting that these proteins hinder the progression of the DNA synthesis at these sites. Overall these results raise the possibility that, upon binding to hairpins formed onto ssDNA during fork progression, the DNA-PK complex interferes with replication fork dynamics in vivo., (© 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Break-induced replication (BIR) is a pathway of homology-directed repair that repairs one-ended DNA breaks, such as those formed at broken replication forks or uncapped telomeres. In contrast to conventional S phase DNA synthesis, BIR proceeds by a migrating D-loop and results in conservative synthesis of the nascent strands. DNA polymerase delta (Pol δ) initiates BIR; however, it is not known whether synthesis of the invading strand switches to a different polymerase or how the complementary strand is synthesized. By using alleles of the replicative DNA polymerases that are permissive for ribonucleotide incorporation, thus generating a signature of their action in the genome that can be identified by hydrolytic end sequencing, we show that Pol δ replicates both the invading and the complementary strand during BIR. In support of this conclusion, we show that depletion of Pol δ from cells reduces BIR, whereas depletion of Pol ε has no effect., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Poly(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., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

DNA methylation is an essential epigenetic mark in mammals that has to be re-established after each round of DNA replication. The protein UHRF1 is essential for this process; it has been proposed that the protein targets newly replicated DNA by cooperatively binding hemi-methylated DNA and H3K9me2/3, but this model leaves a number of questions unanswered. Here, we present evidence for a direct recruitment of UHRF1 by the replication machinery via DNA ligase 1 (LIG1). A histone H3K9-like mimic within LIG1 is methylated by G9a and GLP and, compared with H3K9me2/3, more avidly binds UHRF1. Interaction with methylated LIG1 promotes the recruitment of UHRF1 to DNA replication sites and is required for DNA methylation maintenance. These results further elucidate the function of UHRF1, identify a non-histone target of G9a and GLP, and provide an example of a histone mimic that coordinates DNA replication and DNA methylation maintenance., (Copyright © 2017 Elsevier Inc. All rights reserved.)