XRCC1 Prevents Replication Fork Instability during Misincorporation of the DNA Demethylation Bases 5-Hydroxymethyl-2′-Deoxycytidine and 5-Hydroxymethyl-2′-Deoxyuridine

Whilst avoidance of chemical modifications of DNA bases is essential to maintain genome stability, during evolution eukaryotic cells have evolved a chemically reversible modification of the cytosine base. These dynamic methylation and demethylation reactions on carbon-5 of cytosine regulate several cellular and developmental processes such as embryonic stem cell pluripotency, cell identity, differentiation or tumourgenesis. Whereas these physiological processes are well characterized, very little is known about the toxicity of these cytosine analogues when they incorporate during replication. Here, we report a role of the base excision repair factor XRCC1 in protecting replication fork upon incorporation of 5-hydroxymethyl-2′-deoxycytosine (5hmC) and its deamination product 5-hydroxymethyl-2′-deoxyuridine (5hmU) during DNA synthesis. In the absence of XRCC1, 5hmC exposure leads to increased genomic instability, replication fork impairment and cell lethality. Moreover, the 5hmC deamination product 5hmU recapitulated the genomic instability phenotypes observed by 5hmC exposure, suggesting that 5hmU accounts for the observed by 5hmC exposure. Remarkably, 5hmC-dependent genomic instability and replication fork impairment seen in Xrcc1−/− cells were exacerbated by the trapping of Parp1 on chromatin, indicating that XRCC1 maintains replication fork stability during processing of 5hmC and 5hmU by the base excision repair pathway. Our findings uncover natural epigenetic DNA bases 5hmC and 5hmU as genotoxic nucleosides that threaten replication dynamics and genome integrity in the absence of XRCC1.

Structures of LIG1 engaging with mutagenic mismatches inserted by polβ in base excision repair

DNA ligase I (LIG1) catalyzes final ligation step following DNA polymerase (pol) β gap filling and an incorrect nucleotide insertion by polβ creates a nick repair intermediate with mismatched end at the downstream steps of base excision repair (BER) pathway. Yet, how LIG1 discriminates against the mutagenic 3'-mismatches at atomic resolution remains undefined. Here, we determined X-ray structures of LIG1/nick DNA complexes with G:T and A:C mismatches and uncovered the ligase strategies that favor or deter ligation of base substitution errors. Our structures revealed that LIG1 active site can accommodate G:T mismatch in a similar conformation with A:T base pairing, while it stays in the LIG1-adenylate intermediate during initial step of ligation reaction in the presence of A:C mismatch at 3'-strand. Moreover, we showed mutagenic ligation and aberrant nick sealing of the nick DNA substrates with 3'-preinserted dG:T and dA:C mismatches, respectively. Finally, we demonstrated that AP-Endonuclease 1 (APE1), as a compensatory proofreading enzyme, interacts and coordinates with LIG1 during mismatch removal and DNA ligation. Our overall findings and ligase/nick DNA structures provide the features of accurate versus mutagenic outcomes at the final BER steps where a multi-protein complex including polβ, LIG1, and APE1 can maintain accurate repair.

Structures of LIG1 engaging with mutagenic mismatches inserted by polβ in base excision repair

DNA ligase I (LIG1) catalyzes final ligation step following DNA polymerase (pol) β gap filling and an incorrect nucleotide insertion by polβ creates a nick repair intermediate with mismatched end at the downstream steps of base excision repair (BER) pathway. Yet, how LIG1 discriminates against the mutagenic 3'-mismatches at atomic resolution remains undefined. Here, we determined X-ray structures of LIG1/nick DNA complexes with G:T and A:C mismatches and uncovered the ligase strategies that favor or deter ligation of base substitution errors. Our structures revealed that LIG1 active site can accommodate G:T mismatch in a similar conformation with A:T base pairing, while it stays in the LIG1-adenylate intermediate during initial step of ligation reaction in the presence of A:C mismatch at 3'-strand. Moreover, we showed mutagenic ligation and aberrant nick sealing of the nick DNA substrates with 3'-preinserted dG:T and dA:C mismatches, respectively. Finally, we demonstrated that AP-Endonuclease 1 (APE1), as a compensatory proofreading enzyme, interacts and coordinates with LIG1 during mismatch removal and DNA ligation. Our overall findings and ligase/nick DNA structures provide the features of accurate versus mutagenic outcomes at the final BER steps where a multi-protein complex including polβ, LIG1, and APE1 can maintain accurate repair.

DNA Damage Repair: Predictor of Platinum Efficacy in Ovarian Cancer?

Ovarian cancer (OC) is the seventh most common type of cancer in women worldwide. Treatment for OC usually involves a combination of surgery and chemotherapy with carboplatin and paclitaxel. Platinum-based agents exert their cytotoxic action through development of DNA damage, including the formation of intra- and inter-strand cross-links, as well as single-nucleotide damage of guanine. Although these agents are highly efficient, intrinsic and acquired resistance during treatment are relatively common and remain a major challenge for platinum-based therapy. There is strong evidence to show that the functionality of various DNA repair pathways significantly impacts tumor response to treatment. Various DNA repair molecular components were found deregulated in ovarian cancer, including molecules involved in homologous recombination repair (HRR), nucleotide excision repair (NER), mismatch repair (MMR), non-homologous end-joining (NHEJ), and base excision repair (BER), which can be possibly exploited as novel therapeutic targets and sensitive/effective biomarkers. This review attempts to summarize published data on this subject and thus help in the design of new mechanistic studies to better understand the involvement of the DNA repair in the platinum drugs resistance, as well as to suggest new therapeutic perspectives and potential targets.

Optimization of N-piperidinyl-benzimidazolone derivatives as potent and selective inhibitors of 8-Oxo Guanine DNA Glycosylase 1

8-oxo Guanine DNA Glycosylase 1 is the initiating enzyme within base excision repair and removes oxidized guanines from damaged DNA. Since unrepaired 8-oxoG could lead to G:C→T:A transversion, base removal is of the utmost importance for cells to ensure genomic integrity. For cells with elevat-ed levels of reactive oxygen species this dependency is further increased. In the past we and others have validated OGG1 as a target for inhibitors to treat cancer and inflammation. Here, we present the optimization campaign that led to the broadly used tool compound TH5487. Based on a high-throughput screen, we performed hit to lead expansion and arrived at potent and selective substituted N-piperidinyl-benzimidazolones. Using X-ray crystallography data, we describe the surprising bind-ing mode of the most potent member of the class, TH8535. Here, the N-Piperidinyl-linker adopts a chair instead of a boat conformation which was found for weaker analogues. We further demonstrate cellular target engagement and efficacy of TH8535 against a number of cancer cell lines.

1H NMR chemical exchange techniques reveal local and global effects of oxidized cytosine derivatives

5-carboxycytosine (5caC) is a rare epigenetic modification found in nucleic acids of all domains of life. Despite its sparse genomic abundance, 5caC is presumed to play essential regulatory roles in transcription, maintenance and base-excision processes in DNA. In this work, we utilize nuclear magnetic resonance (NMR) spectroscopy to address the effects of 5caC incorporation into canonical DNA strands at multiple pH and temperature conditions. Our results demonstrate that 5caC has a pH-dependent global destabilizing and a base-pair mobility enhancing local impact on dsDNA, albeit without any detectable influence on the ground-state B-DNA structure. Measurement of hybridization thermodynamics and kinetics of 5caC-bearing DNA duplexes highlighted how acidic environment (pH 5.8 and 4.7) destabilizes the double-stranded structure by ≈10-20 kJ mol-1 at 37 °C when compared to the same sample at neutral pH. Protonation of 5caC results in a lower activation energy for the dissociation process and a higher barrier for annealing. Studies on conformational exchange on the µs time scale regime revealed a sharply localized base-pair motion involving exclusively the modified site and its immediate surroundings. By direct comparison with canonical and 5-formylcytosine (5fC)-edited strands, we were able to address the impact of the two most oxidized naturally occurring cytosine derivatives in the genome. These insights on 5caC's subtle sensitivity to acidic pH contribute to the long standing questions of its capacity as a substrate in base excision repair processes and its purpose as an independent, stable epigenetic mark.

Caught in motion: human NTHL1 undergoes interdomain rearrangement necessary for catalysis

Base excision repair (BER) is the main pathway protecting cells from the continuous damage to DNA inflicted by reactive oxygen species. BER is initiated by DNA glycosylases, each of which repairs a particular class of base damage. NTHL1, a bifunctional DNA glycosylase, possesses both glycolytic and β-lytic activities with a preference for oxidized pyrimidine substrates. Defects in human NTHL1 drive a class of polyposis colorectal cancer. We report the first X-ray crystal structure of hNTHL1, revealing an open conformation not previously observed in the bacterial orthologs. In this conformation, the six-helical barrel domain comprising the helix-hairpin-helix (HhH) DNA binding motif is tipped away from the iron sulphur cluster-containing domain, requiring a conformational change to assemble a catalytic site upon DNA binding. We found that the flexibility of hNTHL1 and its ability to adopt an open configuration can be attributed to an interdomain linker. Swapping the human linker sequence for that of Escherichia coli yielded a protein chimera that crystallized in a closed conformation and had a reduced activity on lesion-containing DNA. This large scale interdomain rearrangement during catalysis is unprecedented for a HhH superfamily DNA glycosylase and provides important insight into the molecular mechanism of hNTHL1.