BACKGROUND:Susceptibility to lung cancer and COPD may reflect interindividual differences in DNA repair capacity (DRC) to complex airborne particulates. METHODS:Lymphocyte DRC was quantified in two high-risk smoker cohorts with an alkaline comet chip assay after exposure to total particulate matter (TPM) from PM2.5, wood smoke (WS), mainstream (MS) and sidestream (SS) cigarette smoke, and H2O2. The micronucleus assay evaluated clastogenic response. TPM composition was profiled, and repair-pathway polygenic risk scores (PRS) were derived from GWAS with PrediXcan validation. RESULTS:DRC after PM2.5 and WS was highly left-skewed; ∼50% of participants showed <5% repair after PM2.5. Poor vs. good DRC was associated with higher odds of lung cancer (PM2.5 OR = 1.8; WS OR = 2.3), and combined PM2.5+WS further increased risk (OR up to 3.5). For COPD, poor DRC after PM2.5,WS, and MS was associated with OR = 2.3-2.6; combined PM2.5+WS + MS yielded OR = 2.5-2.8. Trends for increased micronuclei were consistent for all exposures, but not significant. Composition analyses identified polar acids in PM2.5 (e.g., phthalic acid) and PAH/NPAH compounds in WS (e.g., 4-nitrobiphenyl) that induced micronuclei at relevant doses. Exploratory PRS analyses suggest associations between lower base excision and single strand break repair (BER/SSBR) pathways and reduced predicted DRC to WS or H2O2, with 5/9 gene expression predictions validated by qRT-PCR. CONCLUSIONS:These findings suggest that functional reduced DRC in response to PM2.5 and WS and underlying BER/SSBR pathway variation can identify smokers at elevated risk for lung cancer and COPD. Scalable comet chip DRC phenotyping of cryopreserved lymphocytes may inform risk stratification in regions impacted by air pollution and wildfire smoke.

Eukaryotic genomic DNA is packaged into chromatin through a fundamental repeating unit known as the nucleosome core particle. Within this chromatin context, genomic DNA is constantly exposed to endogenous and exogenous stress that result in the formation of DNA damage, which must be effectively repaired to maintain genome stability. Single-strand breaks (SSBs) are among the most prevalent forms of DNA damage that arise via the oxidation-induced disintegration of the sugar-phosphate backbone or as repair intermediates during base excision repair. DNA ligase IIIα (LigIIIα) is one of the primary enzymes responsible for repairing SSBs containing an intact 5'-phosphate and 3'-OH (nick) during the terminal step of single-strand break repair (SSBR) and base excision repair (BER) pathways. To date, a complete mechanistic description for how LigIIIα processes nicks within chromatin remains elusive. Here, we use a combination of biochemical assays, molecular dynamics simulations, and cryogenic electron microscopy (cryo-EM) to define the molecular basis of nick ligation in the nucleosome by LigIIIα. Quantitative enzyme kinetics reveal that the LigIIIα ligation rate is highly dependent on the translational position of the nick in the nucleosome, where nicks near the nucleosome entry/exit site are ligated with moderate efficiency and nicks near the nucleosome dyad are refractory to ligation. Cryo-EM structures of LigIIIα bound to nicks at four unique translational positions in the nucleosome reveal the structural basis for this position-dependent catalytic activity, identifying that local steric constraints imposed by the histone octamer prevent LigIIIα from readily adopting a ligation-competent conformation. Further biochemical and structural analysis demonstrates that the scaffolding protein XRCC1, which forms a heterodimer with LigIIIα, does not substantially alter the ability of LigIIIα to bind or ligate nicks in the nucleosome. Together, this work provides foundational insight into the processing of nicks in the nucleosome during the terminal step of SSBR/BER.

Poly-ADP-ribose polymerases 1 and 2 (PARP1 and 2) are critical sensors of DNA-strand breaks and targets for cancer therapy. Upon DNA damage, PARP1 and 2 synthesize poly-ADP-ribose (PAR) chains on themselves and other substrates, facilitating DNA single-strand break repair by recruiting PAR-binding DNA repair factors, including X-ray repair cross-complementing group 1 (XRCC1) and aprataxin and polynucleotide kinase phosphatase-like factor (APLF). While diverse DNA lesions activate PARP1, PARP2 is selectively activated by 5' phosphorylated nicks. They function independently and compensate for each other. Previous studies suggest that PARP1 and its PAR chains act upstream to recruit PARP2 to DNA damage sites. Here, we report that the scaffold protein XRCC1 mediates PARP1- and PAR-dependent recruitment of PARP2 to damage sites. XRCC1-deficiency causes hyperactivation of PARP1 while attenuating micro-irradiation-induced PARP2 foci. Mechanistically, the BRCT1 domain of XRCC1 binds to PAR, while its BRCT2 domain interacts with the PARP2 catalytic domain independently of the PARP2 enzymatic activity and the LIG3 BRCT domain via residues D575 and Y576. This mode of PARP2 enrichment is important for the recruitment of certain PAR-binding proteins, such as APLF, but dispensable for others, such as the XRCC1-BRCT1 domain. These findings highlight the distinct role of PARP1 and PARP2 in PAR synthesis and uncover unexpected hierarchical roles of PARP1 and XRCC1 upstream of PARP2.

Non-homologous end joining (NHEJ) is a major pathway of DNA double strand break (DSB) repair, capable of directly joining both damaged strands of DNA through the coordinated activities of repair factors that detect the termini, physically bridge them together, and perform the chemistry necessary to complete repair. NHEJ is capable of repairing a variety of damaged DNA, employing various accessory end-processing factors to resolve chemically blocked ends, trim overhangs, and fill gaps in order to achieve directly ligatable DNA ends. To investigate the molecular mechanisms underlying end-processing, we determined the cryo-EM structure of the NHEJ specific polymerase Pol λ bound to the short-range synaptic complex, uncovering the mode of its recruitment to the complex as well as a putative model for its activity. Furthermore, the coordinated end-processing activities of the short-range (SR) synaptic complex simultaneously bound by both Pol λ and PNKP, another accessory factor, demonstrates the ability of NHEJ to form large, multifunctional repair complexes capable of processing a variety of different DNA end structures to effect repair.

Ligation of DNA single strand breaks is critical for maintaining genome integrity during DNA replication and repair. DNA Ligase III (LIG3α) forms an important complex with X-ray cross complementing protein 1 (XRCC1) during single strand break and base excision repair. We utilized a real time single molecule approach to quantify DNA binding kinetics of Halo-tagged LIG3α and XRCC1-YFP from nuclear extracts on long DNA substrates containing nicks, nucleosomes or nicks embedded in nucleosomes. LIG3α displayed higher affinity for nicks than XRCC1 with the LIG3α catalytic core and N-terminal zinc finger (ZnF) competing for nick engagement. Surprisingly, compared to single strand breaks in naked DNA, LIG3α bound even more avidly to an undamaged nucleosome reconstituted on the 601-sequence, with binding dependent on two arginine residues in the N-terminal ZnF. These studies reveal insights into nick detection and identify the role of a novel arginine anchor in LIG3α for engaging nucleosomes.

The joining of DNA single- and double-strand breaks (SSB and DSB) is essential for maintaining genome stability and integrity. While this is ultimately accomplished in human cells by the DNA ligases encoded by the LIG1, LIG3 and LIG4 genes, these enzymes are recruited to DNA breaks through specific interactions with proteins involved in break sensing and recognition and/or break processing. In this review, we focus on the interplay between the DNA break-activated poly (ADP-ribose) polymerases, PARP1 and PARP2, poly (ADP-ribose) (PAR) and the DNA ligases in DNA replication and repair. The most extensively studied example of this interplay is the recruitment of DNA ligase IIIα (LigIIIα) and other repair proteins to SSBs through an interaction between XRCC1, a scaffold protein and partner protein of nuclear LigIIIα, and PAR synthesized by PARP1 and to a lesser extent PARP2. Recently, these proteins have been implicated in a back-up pathway for joining Okazaki fragments that appears to have a critical function even in cells with no defect in the major LigI-dependent pathway. Finally, we discuss the effects of FDA-approved PARP1/2 inhibitors on DNA replication and repair in cancer and non-malignant cells and the potential utility of DNA ligase inhibitors as cancer therapeutics.

This study establishes the physiological role of Fused in Sarcoma (FUS) in mitochondrial DNA (mtDNA) repair and highlights its implications to the pathogenesis of FUS-associated neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). Endogenous FUS interacts with and recruits mtDNA Ligase IIIα (mtLig3) to DNA damage sites within mitochondria, a relationship essential for maintaining mtDNA repair and integrity in healthy cells. Using ALS patient-derived FUS mutant cell lines, a transgenic mouse model, and human autopsy samples, we discovered that compromised FUS functionality hinders mtLig3’s repair role, resulting in increased mtDNA damage and mutations. These alterations cause various manifestations of mitochondrial dysfunction, particularly under stress conditions relevant to disease pathology. Importantly, rectifying FUS mutations in patient-derived induced pluripotent cells (iPSCs) preserves mtDNA integrity. Similarly, targeted introduction of human DNA Ligase 1 restores repair mechanisms and mitochondrial activity in FUS mutant cells, suggesting a potential therapeutic approach. Our findings unveil FUS’s critical role in mitochondrial health and mtDNA repair, offering valuable insights into the mechanisms underlying mitochondrial dysfunction in FUS-associated motor neuron disease.

Summary PARP1&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&2 at DNA lesions. Here, we report that unlike Parp2 -/- mice, which develop normally, mice expressing catalytically-inactive Parp2 (E534A, 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) between Okazaki fragments, typically resolved by Lig1. Inactive PARP2, but not its active form or absence, impedes Lig1- and Lig3-mediated ligation, causing dose-dependent replication fork collapse, particularly harmful to erythroblasts with ultra-fast forks. This PARylation-dependent structural function of PARP2 at 5’p-nicks explains the detrimental effects of PARP2 inhibition on erythropoiesis, revealing the mechanism behind the PARPi-induced anemia and leukemia, especially those with TP53/CHK2 loss. Significance This work shows that the hematological toxicities associated with PARP inhibitors stem not from impaired PARP1 or PARP2 enzymatic activity but rather from the presence of inactive PARP2 protein. Mechanistically, these toxicities reflect a unique role of PARP2 at 5’-phosphorylated DNA nicks during DNA replication in erythroblasts.

DNA damage is a fundamental molecular cause of genomic instability. Base excision repair (BER) is one line of defense to minimize the potential mutagenicity and/or toxicity derived from damaged nucleobase lesions. However, BER in the context of chromatin, in which eukaryotic genomic DNA is compacted through a hierarchy of DNA-histone protein interactions, is not fully understood. Here, we investigate the activity of BER enzymes at 27 unique geometric locations in a nucleosome core particle (NCP), which is the minimal unit of packaging in chromatin. The BER enzymes include uracil DNA glycosylase (UDG), AP endonuclease 1 (APE1), DNA polymerase β (Pol β), and DNA ligase IIIα complexed with X-ray repair cross complementing group 1 (LigIIIα/XRCC1). This global analysis of BER reveals that initiation of the repair event by UDG is dictated by the rotational position of the lesion. APE1 has robust activity at locations where repair is initiated whereas the repair event stalls at the Pol β nucleotide incorporation step within the central ∼45 bp of nucleosomal DNA. The final step of the repair, catalyzed by LigIIIα/XRCC1, is achieved only in the entry/exit regions of the NCP when nick sites are transiently exposed by unwrapping from the histones. Kinetic assays further elucidate that the location of the damaged lesion modulates enzymatic activity. Notably, these data indicate that some of the BER enzymes can act at a significant number of locations even in the absence of chromatin remodelers or other cellular factors. These results inform genome wide maps of DNA damage and mutations and contribute to our understanding of mutational hotspots and signatures.

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 joining of breaks in the DNA phosphodiester backbone is essential for genome integrity. Breaks are generated during normal processes such as DNA replication, cytosine demethylation during differentiation, gene rearrangement in the immune system and germ cell development. In addition, they are generated either directly by a DNA damaging agent or indirectly due to damage excision during repair. Breaks are joined by a DNA ligase that catalyzes phosphodiester bond formation at DNA nicks with 30 hydroxyl and 50 phosphate termini. Three human genes encode ATP-dependent DNA ligases. These enzymes have a conserved catalytic core consisting of three subdomains that encircle nicked duplex DNA during ligation. The DNA ligases are targeted to different nuclear DNA transactions by specific protein-protein interactions. Both DNA ligase IIIa and DNA ligase IV form stable complexes with DNA repair proteins, XRCC1 and XRCC4, respectively. There is functional redundancy between DNA ligase I and DNA ligase IIIa in DNA replication, excision repair and single-strand break repair. Although DNA ligase IV is a core component of the major double-strand break repair pathway, non-homologous end joining, the other enzymes participate in minor, alternative double-strand break repair pathways. In contrast to the nucleus, only DNA ligase IIIa is present in mitochondria and is essential for maintaining the mitochondrial genome. Human immunodeficiency syndromes caused by mutations in either LIG1 or LIG4 have been described. Preclinical studies with DNA ligase inhibitors have identified potentially targetable abnormalities in cancer cells and evidence that DNA ligases are potential targets for cancer therapy. (c) 2023 Elsevier Ltd. All rights reserved.

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.

Supplement Figure S1. Chemical Structures of Letrozole, Anastrozole, and Exemestane

Panel A. Characterization of HeLA cells expressing mitochondrially-targeted E. coli DNA ligase. DNA encoding the E. coli DNA detected by PCR and expression of GFP-tagged E. coli DNA ligase detected by immunoblotting. Panel B. Effect of L67 on DNA ligation by purified DN Ligase IIIalpha/XRCC1 and E.coli DNA ligase

Efficient repair of oxidized DNA is critical for genome-integrity maintenance. Cockayne syndrome protein B (CSB) is an ATP-dependent chromatin remodeler that collaborates with Poly(ADP-ribose) polymerase I (PARP1) in the repair of oxidative DNA lesions. How these proteins integrate during DNA repair remains largely unknown. Here, using chromatin co-fractionation studies, we demonstrate that PARP1 and PARP2 promote recruitment of CSB to oxidatively-damaged DNA. CSB, in turn, contributes to the recruitment of XRCC1, and histone PARylation factor 1 (HPF1), and promotes histone PARylation. Using alkaline comet assays to monitor DNA repair, we found that CSB regulates single-strand break repair (SSBR) mediated by PARP1 and PARP2. Strikingly, CSB's function in SSBR is largely bypassed when transcription is inhibited, suggesting CSB-mediated SSBR occurs primarily at actively transcribed DNA regions. While PARP1 repairs SSBs at sites regardless of the transcription status, we found that PARP2 predominantly functions in actively transcribed DNA regions. Therefore, our study raises the hypothesis that SSBR is executed by different mechanisms based on the transcription status.

DNA double-strand breaks (DSBs) are a highly cytotoxic form of DNA damage and the incorrect repair of DSBs is linked to carcinogenesis. The conserved error-prone non-homologous end joining (NHEJ) pathway has a key role in determining the effects of DSB-inducing agents that are used to treat cancer as well as the generation of the diversity in antibodies and T cell receptors. Using single-particle cryo-electron microscopy, we visualized key DNA-protein complexes that are formed by human NHEJ factors. A near-complete NHEJ reaction cycle and a life cycle of DNA-PKcs (catalytic subunit of the DNA-dependent protein kinase) are proposed from the models. The Ku70/80 heterodimer (Ku), DNA-PKcs, XRCC4 and XLF first form a long-range synaptic complex in which the DNA ends are held approximately 115 Å apart. Two DNA end-bound subcomplexes comprising Ku and DNA-PKcs are linked by interactions between the DNA-PKcs subunits and a scaffold comprising LigIV, XRCC4, and XLF. The relative orientation of the DNA-PKcs molecules suggests a mechanism for autophosphorylation in trans, which leads to substantial outward rotation of both Ku and DNA-PKcs to promote DNA double-strand break exposure and DNA-PKcs dissociation. Upon DNA-PKcs dissociation, the long-range synaptic complex transit into the short-range synaptic complex, within which the Ku-bound DNA ends are aligned for processing and ligation by the XLF-anchored scaffold, and a single catalytic domain of LigIV is stably associated with a nick between the two Ku molecules, suggesting that the joining of both strands of a DSB involves both LigIV molecules. In addition, we captured a dimeric state of catalytically inactive DNA-PKcs, which resembles previously determined structures of PIKK family kinases, revealing a model of the full life cycle of DNA-PKcs during NHEJ.

Supplementary Figure 4 from Rational Design of Human DNA Ligase Inhibitors that Target Cellular DNA Replication and Repair

Panel A. Induction of gammaH2AX foci in HeLa cells, HeLa Rho minus cells and HeLa cells expressing mitochondrially targeted E. coli DNA ligase by L67. Panel B. N-acetyl cysteine inhibits L67-induced autophosphorylation of ATM in HeLa cells.