DNA Repair最新文献

Epigenetic cytosine methylation covers most of genomic CpG dinucleotides in human cells. In addition to common deamination-mediated mutagenesis at CpG sites, an alternative deamination-independent pathway associated with DNA polymerase activity was previously described. This mutagenesis is characterized by the TCG→TTG mutational signature and is believed to arise from dAMP misincorporation opposite 5-methylcytosine (mC) or its oxidized derivative 5-hydroxymethylcytosine (hmC) by B-family replicative DNA polymerases with disrupted proofreading 3→5′-exonuclease activity. In addition to being less stable and pro-mutagenic themselves, cytosine modifications also increase the risk of adjacent nucleotides damage, including the formation of 8-oxo-2'-deoxyguanosine (8-oxoG), a well-known mutagenic lesion. The effect of cytosine methylation on error-prone DNA polymerases lacking proofreading activity and involved in repair and DNA translesion synthesis remains unexplored. Here we analyze the efficiency and fidelity of translesion Y-family polymerases (Pol κ, Pol η, Pol ι and REV1) and primase-polymerase PrimPol opposite mC and hmC as well as opposite 8-oxoG adjacent to mC in the TCG context. We demonstrate that epigenetic cytosine modifications suppress Pol ι and REV1 activities and lead to increasing dAMP misincorporation by PrimPol, Pol κ and Pol ι in vitro. Cytosine methylation also increases misincorporation of dAMP opposite the adjacent 8-oxoG by PrimPol, decreases the TLS activity of Pol η opposite the lesion but increases dCMP incorporation opposite 8-oxoG by REV1. Altogether, these data suggest that methylation and hydroxymethylation of cytosine alter activity and fidelity of translesion DNA polymerases.

The KU heterodimer (KU70/80) is rapidly recruited to DNA double-strand breaks (DSBs) to regulate their processing and repair. Previous work has revealed that the amino-terminal von Willebrand-like (vWA-like) domain in KU80 harbours a conserved hydrophobic pocket that interacts with a short peptide motif known as the Ku-binding motif (KBM). The KBM is present in a variety of DNA repair proteins such as APLF, CYREN, and Werner protein (WRN). Here, to investigate the importance of KBM-mediated protein-protein interactions for KU80 function, we employed KU80-deficient Chinese Hamster Ovary (Xrs-6) cells transfected with RFP-tagged wild-type human KU80 or KU80 harbouring a mutant vWA-like domain (KU80^L68R). Surprisingly, while mutant RFP-KU80^L68R largely or entirely restored NHEJ efficiency and radiation resistance in KU80-deficient Xrs-6 cells, it failed to restore cellular resistance to DNA replication stress induced by camptothecin (CPT) or hydroxyurea (HU). Moreover, KU80-deficient Xrs-6 cells expressing RFP-KU80^L68R accumulated pan-nuclear γH2AX in an S/G2-phase-dependent manner following treatment with CPT or HU, suggesting that the binding of KU80 to one or more KBM-containing proteins is required for the processing and/or repair of DNA ends that arise during DNA replication stress. Consistent with this idea, depletion of WRN helicase/exonuclease recapitulated the CPT-induced γH2AX phenotype, and did so epistatically with mutation of the KU80 vWA-like domain. These data identify a role for the KBM-binding by KU80 in the response and resistance of CHO cells to arrested and/or collapsed DNA replication forks, and implicate the KBM-mediated interaction of KU80 with WRN as a critical effector of this role.

The maintenance of genome integrity by DNA damage response machineries is key to protect cells against pathological development. In cell nuclei, these genome maintenance machineries operate in the context of chromatin, where the DNA wraps around histone proteins. Here, we review recent findings illustrating how the chromatin substrate modulates genome maintenance mechanisms, focusing on the regulatory role of histone variants and post-translational modifications. In particular, we discuss how the pre-existing chromatin landscape impacts DNA damage formation and guides DNA repair pathway choice, and how DNA damage-induced chromatin alterations control DNA damage signaling and repair, and DNA damage segregation through cell divisions. We also highlight that pathological alterations of histone proteins may trigger genome instability by impairing chromosome segregation and DNA repair, thus defining new oncogenic mechanisms and opening up therapeutic options.

To identify new molecular components of the Brh2-governed homologous recombination (HR)-network in the highly radiation-resistant fungus Ustilago maydis, we undertook a genetic screen for suppressors of blm-^KR hydroxyurea (HU)-sensitivity. Twenty DNA-damage sensitive mutants were obtained, three of which showing slow-growth phenotypes. Focusing on the “normally” growing candidates we identified five mutations, two in previously well-defined genes (Rec2 and Rad51) and the remaining three in completely uncharacterized genes (named Rec3, Bls9 and Zdr1). A common feature among these novel factors is their prominent role in DNA repair. Rec3 contains the P-loop NTPase domain which is most similar to that found in U. maydis Rec2 protein, and like Rec2, Rec3 plays critical roles in induced allelic recombination, is crucial for completion of meiosis, and with regard to DNA repair Δrec3 and Δrec2 are epistatic to one another. Importantly, overexpression of Brh2 in Δrec3 can effectively restore DNA-damage resistance, indicating a close functional connection between Brh2 and Rec3. The Bls9 does not seem to have any convincing domains that would give a clue as to its function. Nevertheless, we present evidence that, besides being involved in DNA-repair, Bls9 is also necessary for HR between chromosome homologs. Moreover, Δbls9 showed epistasis with Δbrh2 with respect to killing by DNA-damaging agents. Both, Rec3 and Bls9, play an important role in protecting the genome from mutations. Zdr1 is Cys2-His2 zinc finger (C2H2-ZF) protein, whose loss does not cause a detectable change in HR. Also, the functions of both Bls9 and Zdr1 genes are dispensable in meiosis and sporulation. However, Zdr1 appears to have overlapping activities with Blm and Mus81 in protecting the organism from methyl methanesulfonate- and diepoxybutane-induced DNA-damage. Finally, while deletion of Rec3 and Zdr1 can suppress HU-sensitivity of blm-^KR, Δgen1, and Δmus81 mutants, interestingly loss of Bls9 does not rescue HU-sensitivity of Δgen1.

FANCM is a multifunctional DNA repair enzyme that acts as a sensor and coordinator of replication stress responses, especially interstrand crosslink (ICL) repair mediated by the Fanconi anaemia (FA) pathway. Its specialised ability to bind and remodel branched DNA structures enables diverse genome maintenance activities. Through ATP-powered “branchpoint translocation”, FANCM can promote fork reversal, facilitate replication traverse of ICLs, resolve deleterious R-loop structures, and restrain recombination. These remodelling functions also support a role as sensor of perturbed replication, eliciting checkpoint signalling and recruitment of downstream repair factors like the Fanconi anaemia FANCI:FANCD2 complex. Accordingly, FANCM deficiency causes chromosome fragility and cancer susceptibility. Other recent advances link FANCM to roles in gene editing efficiency and meiotic recombination, along with emerging synthetic lethal relationships, and targeting opportunities in ALT-positive cancers. Here we review key properties of FANCM's biochemical activities, with a particular focus on branchpoint translocation as a distinguishing characteristic.

Mutations in isocitrate dehydrogenase isoform 1 (IDH1) are primarily found in secondary glioblastoma (GBM) and low-grade glioma but are rare in primary GBM. The standard treatment for GBM includes radiation combined with temozolomide, an alkylating agent. Fortunately, IDH1 mutant gliomas are sensitive to this treatment, resulting in a more favorable prognosis. However, it’s estimated that up to 75 % of IDH1 mutant gliomas will progress to WHO grade IV over time and develop resistance to alkylating agents. Therefore, understanding the mechanism(s) by which IDH1 mutant gliomas confer sensitivity to alkylating agents is crucial for developing targeted chemotherapeutic approaches. The base excision repair (BER) pathway is responsible for repairing most base damage induced by alkylating agents. Defects in this pathway can lead to hypersensitivity to these agents due to unresolved DNA damage. The coordinated assembly and disassembly of BER protein complexes are essential for cell survival and for maintaining genomic integrity following alkylating agent exposure. These complexes rely on poly-ADP-ribose formation, an NAD⁺-dependent post-translational modification synthesized by PARP1 and PARP2 during the BER process. At the lesion site, poly-ADP-ribose facilitates the recruitment of XRCC1. This scaffold protein helps assemble BER proteins like DNA polymerase beta (Polβ), a bifunctional DNA polymerase containing both DNA synthesis and 5′-deoxyribose-phosphate lyase (5’dRP lyase) activity. Here, we confirm that IDH1 mutant glioma cells have defective NAD⁺ metabolism, but still produce sufficient nuclear NAD⁺ for robust PARP1 activation and BER complex formation in response to DNA damage. However, the overproduction of 2-hydroxyglutarate, an oncometabolite produced by the IDH1 R132H mutant protein, suppresses BER capacity by reducing Polβ protein levels. This defines a novel mechanism by which the IDH1 mutation in gliomas confers cellular sensitivity to alkylating agents and to inhibitors of the poly-ADP-ribose glycohydrolase, PARG.

Neurodegenerative disorders are primarily characterized by neuron loss progressively leading to cognitive decline and the manifestation of incurable and debilitating conditions, such as Alzheimer's, Parkinson's, and Huntington's diseases. Loss of genome maintenance causally contributes to age-related neurodegeneration, as exemplified by the premature appearance of neurodegenerative features in a growing family of human syndromes and mice harbouring inborn defects in DNA repair. Here, we discuss the relevance of persistent DNA damage, key DNA repair mechanisms and compromised genome integrity in age-related neurodegeneration highlighting the significance of investigating these connections to pave the way for the development of rationalized intervention strategies aimed at delaying the onset of neurodegenerative disorders and promoting healthy aging.

Tonicity-responsive enhancer binding protein (TonEBP) is a stress-responsive protein that plays a critical role in the regulation of gene expression and cellular adaptation to stressful environments. Recent studies uncovered the novel role of TonEBP in the DNA damage response, which significantly impacts genomic stability. This review provides a comprehensive overview of the novel role of TonEBP in DNA damage repair, including its involvement in the DNA damage bypass pathway and the recognition and resolution of DNA damage-induced R-loop structures.

DNA double-strand breaks (DSBs) represent one of the most severe threats to genomic integrity, demanding intricate repair mechanisms within eukaryotic cells. A diverse array of factors orchestrates the complex choreography of DSB signaling and repair, encompassing repair pathways, such as non-homologous end-joining, homologous recombination, and polymerase-θ-mediated end-joining. This review looks into the intricate decision-making processes guiding eukaryotic cells towards a particular repair pathway, particularly emphasizing the processing of two-ended DSBs. Furthermore, we elucidate the transformative role of Cas9, a site-specific endonuclease, in revolutionizing our comprehension of DNA DSB repair dynamics. Additionally, we explore the burgeoning potential of Cas9's remarkable ability to induce sequence-specific DSBs, offering a promising avenue for precise targeting of tumor cells. Through this comprehensive exploration, we unravel the intricate molecular mechanisms of cellular responses to DSBs, shedding light on both fundamental repair processes and cutting-edge therapeutic strategies.

DNA strand breaks activate Poly(ADP-ribose) polymerase (PARP) 1 and 2, which use NAD+ as the substrate to covalently conjugate ADP-ribose on themselves and other proteins (e.g., Histone) to promote chromatin relaxation and recruit additional DNA repair factors. Enzymatic inhibitors of PARP1 and PARP2 (PARPi) are promising cancer therapy agents that selectively target BRCA1- or BRCA2- deficient cancers. As immediate early responders to DNA strand breaks with robust activities, PARP1 and PARP2 normally form transient foci (<10 minutes) at the micro-irradiation-induced DNA lesions. In addition to enzymatic inhibition, PARPi also extend the presence of PARP1 and PARP2 at DNA lesions, including at replication forks, where they may post a physical block for subsequent repair and DNA replication. The dynamic nature of PARP1 and PARP2 foci made live cell imaging a unique platform to detect subtle changes and the functional interaction among PARP1, PARP2, and their regulators. Recent imaging studies have provided new understandings of the biological consequence of PARP inhibition and uncovered functional interactions between PARP1 and PARP2 and new regulators (e.g., histone poly(ADP-ribosylation) factor). Here, we review recent advances in dissecting the temporal and spatial Regulation of PARP1 and PARP2 at DNA lesions and discuss their physiological implications on both cancer and normal cells.