Poly(ADP-ribose) polymerases (PARPs) facilitate the repair of DNA single-strand breaks (SSBs). When PARPs are inhibited, unrepaired SSBs colliding with replication forks give rise to cytotoxic ...double-strand breaks. These are normally rescued by homologous recombination (HR), but, in cells with suboptimal HR, PARP inhibition leads to genomic instability and cell death, a phenomenon currently exploited in the therapy of ovarian cancers in BRCA1/2 mutation carriers. In spite of their promise, resistance to PARP inhibitors (PARPis) has already emerged. In order to identify the possible underlying causes of the resistance, we set out to identify the endogenous source of DNA damage that activates PARPs. We argued that if the toxicity of PARPis is indeed caused by unrepaired SSBs, these breaks must arise spontaneously, because PARPis are used as single agents. We now show that a significant contributor to PARPi toxicity is oxygen metabolism. While BRCA1-depleted or -mutated cells were hypersensitive to the clinically approved PARPi olaparib, its toxicity was significantly attenuated by depletion of OGG1 or MYH DNA glycosylases, as well as by treatment with reactive oxygen species scavengers, growth under hypoxic conditions or chemical OGG1 inhibition. Thus, clinical resistance to PARPi therapy may emerge simply through reduced efficiency of oxidative damage repair.
The mechanisms that underpin how insertions or deletions (indels) become fixed in DNA have primarily been ascribed to replication-related and/or double-strand break (DSB)-related processes. Here, we ...introduce a method to evaluate indels, orientating them relative to gene transcription. In so doing, we reveal a number of surprising findings: First, there is a transcriptional strand asymmetry in the distribution of mononucleotide repeat tracts in the reference human genome. Second, there is a strong transcriptional strand asymmetry of indels across 2,575 whole genome sequenced human cancers. We suggest that this is due to the activity of transcription-coupled nucleotide excision repair (TC-NER). Furthermore, TC-NER interacts with mismatch repair (MMR) under physiological conditions to produce strand bias. Finally, we show how insertions and deletions differ in their dependencies on these repair pathways. Our analytical approach reveals insights into the contribution of DNA repair towards indel mutagenesis in human cells.
A considerable surge of interest in the mismatch repair (MMR) system has been brought about by the discovery of a link between Lynch syndrome, an inherited predisposition to cancer of the colon and ...other organs, and malfunction of this key DNA metabolic pathway. This review focuses on recent advances in our understanding of the molecular mechanisms of canonical MMR, which improves replication fidelity by removing misincorporated nucleotides from the nascent DNA strand. We also discuss the involvement of MMR proteins in two other processes: trinucleotide repeat expansion and antibody maturation, in which MMR proteins are required for mutagenesis rather than for its prevention.
O(6)-Methylguanine ((Me)G) is a highly cytotoxic DNA modification generated by S(N)1-type methylating agents. Despite numerous studies implicating DNA replication, mismatch repair (MMR), and ...homologous recombination (HR) in (Me)G toxicity, its mode of action has remained elusive. We studied the molecular transactions in the DNA of yeast and mammalian cells treated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Although replication fork progression was unaffected in the first cell cycle after treatment, electron microscopic analysis revealed an accumulation of (Me)G- and MMR-dependent single-stranded DNA (ssDNA) gaps in newly replicated DNA. Progression into the second cell cycle required HR, while the following G(2) arrest required the continued presence of (Me)G. Yeast cells overcame this block, while mammalian cells generally failed to recover, and those that did contained multiple sister chromatid exchanges. Notably, the arrest could be abolished by removal of (Me)G after the first S phase. These new data provide compelling support for the hypothesis that MMR attempts to correct (Me)G/C or (Me)G/T mispairs arising during replication. Due to the persistence of (Me)G in the exposed template strand, repair synthesis cannot take place, which leaves single-stranded gaps behind the replication fork. During the subsequent S phase, these gaps cause replication fork collapse and elicit recombination and cell cycle arrest.
Thymine DNA glycosylase (TDG) is a member of the uracil DNA glycosylase (UDG) superfamily of DNA repair enzymes. Owing to its ability to excise thymine when mispaired with guanine, it was proposed to ...act against the mutability of 5-methylcytosine (5-mC) deamination in mammalian DNA. However, TDG was also found to interact with transcription factors, histone acetyltransferases and de novo DNA methyltransferases, and it has been associated with DNA demethylation in gene promoters following activation of transcription, altogether implicating an engagement in gene regulation rather than DNA repair. Here we use a mouse genetic approach to determine the biological function of this multifaceted DNA repair enzyme. We find that, unlike other DNA glycosylases, TDG is essential for embryonic development, and that this phenotype is associated with epigenetic aberrations affecting the expression of developmental genes. Fibroblasts derived from Tdg null embryos (mouse embryonic fibroblasts, MEFs) show impaired gene regulation, coincident with imbalanced histone modification and CpG methylation at promoters of affected genes. TDG associates with the promoters of such genes both in fibroblasts and in embryonic stem cells (ESCs), but epigenetic aberrations only appear upon cell lineage commitment. We show that TDG contributes to the maintenance of active and bivalent chromatin throughout cell differentiation, facilitating a proper assembly of chromatin-modifying complexes and initiating base excision repair to counter aberrant de novo methylation. We thus conclude that TDG-dependent DNA repair has evolved to provide epigenetic stability in lineage committed cells.
Postreplicative mismatch repair Jiricny, Josef
Cold Spring Harbor perspectives in biology,
2013-Apr-01, 2013-04-01, 20130401, Letnik:
5, Številka:
4
Journal Article
Recenzirano
Odprti dostop
The mismatch repair (MMR) system detects non-Watson-Crick base pairs and strand misalignments arising during DNA replication and mediates their removal by catalyzing excision of the ...mispair-containing tract of nascent DNA and its error-free resynthesis. In this way, MMR improves the fidelity of replication by several orders of magnitude. It also addresses mispairs and strand misalignments arising during recombination and prevents synapses between nonidentical DNA sequences. Unsurprisingly, MMR malfunction brings about genomic instability that leads to cancer in mammals. But MMR proteins have recently been implicated also in other processes of DNA metabolism, such as DNA damage signaling, antibody diversification, and repair of interstrand cross-links and oxidative DNA damage, in which their functions remain to be elucidated. This article reviews the progress in our understanding of the mechanism of replication error repair made during the past decade.
Cytotoxicity of cisplatin and mitomycin C (MMC) is ascribed largely to their ability to generate interstrand crosslinks (ICLs) in DNA, which block the progression of replication forks. The processing ...of ICLs requires the
Fanconi anemia (FA) pathway, excision repair, and translesion DNA synthesis (TLS). It also requires homologous recombination (HR), which repairs double-strand breaks (DSBs) generated by cleavage of the blocked replication forks. Here we describe KIAA1018, an evolutionarily conserved protein that has an N-terminal ubiquitin-binding zinc finger (UBZ) and a C-terminal nuclease domain. KIAA1018 is a 5′→3′ exonuclease and a structure-specific endonuclease that preferentially incises 5′ flaps. Like cells from FA patients, human cells depleted of KIAA1018 are sensitized to ICL-inducing agents and display chromosomal instability. The link of KIAA1018 to the FA pathway is further strengthened by its recruitment to DNA damage through interaction of its UBZ domain with monoubiquitylated FANCD2. We therefore propose to name KIAA1018 FANCD2-associated nuclease, FAN1.
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► KIAA1018/FAN1 is an endonuclease with a preference for 5′ flap substrates ► It is also an exonuclease with a 5′→3′ directionality ► KIAA1018/FAN1 deficiency sensitizes cells to cisplatin and mitomycin C ► It is recruited to sites of DNA damage by monoubiquitylated FANCD2
By removing biosynthetic errors from newly synthesized DNA, mismatch repair (MMR) improves the fidelity of DNA replication by several orders of magnitude. Loss of MMR brings about a mutator ...phenotype, which causes a predisposition to cancer. But MMR status also affects meiotic and mitotic recombination, DNA-damage signalling, apoptosis and cell-type-specific processes such as class-switch recombination, somatic hypermutation and triplet-repeat expansion. This article reviews our current understanding of this multifaceted DNA-repair system in human cells.
Interstrand cross-links (ICLs) block replication and transcription and thus are highly cytotoxic. In higher eukaryotes, ICLs processing involves the Fanconi anemia (FA) pathway and homologous ...recombination. Stalled replication forks activate the eight-subunit FA core complex, which ubiquitylates FANCD2-FANCI. Once it is posttranslationally modified, this heterodimer recruits downstream members of the ICL repairosome, including the FAN1 nuclease. However, ICL processing has been shown to also involve MUS81-EME1 and XPF-ERCC1, nucleases known to interact with SLX4, a docking protein that also can bind another nuclease, SLX1. To investigate the role of SLX4 more closely, we disrupted the SLX4 gene in avian DT40 cells. SLX4 deficiency caused cell death associated with extensive chromosomal aberrations, including a significant fraction of isochromatid-type breaks, with sister chromatids broken at the same site. SLX4 thus appears to play an essential role in cell proliferation, probably by promoting the resolution of interchromatid homologous recombination intermediates. Because ubiquitylation plays a key role in the FA pathway, and because the N-terminal region of SLX4 contains a ubiquitin-binding zinc finger (UBZ) domain, we asked whether this domain is required for ICL processing. We found that SLX4â»/â» cells expressing UBZ-deficient SLX4 were selectively sensitive to ICL-inducing agents, and that the UBZ domain was required for interaction of SLX4 with ubiquitylated FANCD2 and for its recruitment to DNA-damage foci generated by ICL-inducing agents. Our findings thus suggest that ubiquitylated FANCD2 recruits SLX4 to DNA damage sites, where it mediates the resolution of recombination intermediates generated during the processing of ICLs.
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