DNA Repair最新文献

Nucleotide excision repair (NER) clears genomes of DNA adducts formed by UV light, environmental agents, and antitumor drugs. Gene mutations that lead to defects in the core NER reaction cause the skin cancer-prone disease xeroderma pigmentosum. In NER, DNA lesions are excised within an oligonucleotide of 25–30 residues via a complex, multi-step reaction that is regulated by protein-protein interactions. These interactions were first characterized in the 1990s using pull-down, co-IP and yeast two-hybrid assays. More recently, high-resolution structures and detailed functional studies have started to yield detailed pictures of the progression along the NER reaction coordinate. In this review, we highlight how the study of interactions among proteins by structural and/or functional studies have provided insights into the mechanisms by which the NER machinery recognizes and excises DNA lesions. Furthermore, we identify reported, but poorly characterized or unsubstantiated interactions in need of further validation.

Given its central role in life, DNA is remarkably easy to damage. Double strand breaks (DSBs) are the most toxic form of DNA damage, and DSBs pose the greatest danger to genomic integrity. In higher vertebrates, the non-homologous end joining pathway (NHEJ) is the predominate pathway that repairs DSBs. NHEJ has three steps: 1) DNA end recognition by the DNA dependent protein kinase [DNA-PK], 2) DNA end-processing by numerous NHEJ accessory factors, and 3) DNA end ligation by the DNA ligase IV complex (LX4). Although this would appear to be a relatively simple mechanism, it has become increasingly apparent that it is not.

Recently, much insight has been derived regarding the mechanism of non-homologous end joining through a proliferation of cryo-EM studies, structure-function mutational experiments informed by these new structural data, and novel single-molecule imaging approaches. An emerging consensus in the field is that NHEJ progresses from initial DSB end recognition by DNA-PK to synapsis of the two DNA ends in a long-range synaptic complex where ends are held too far apart (115 Å) for ligation, and then progress to a short-range synaptic complex where ends are positioned close enough for ligation. What was surprising from these structural studies was the observation of two distinct types of DNA-PK dimers that represent NHEJ long-range complexes. In this review, we summarize current knowledge about the function of the distinct NHEJ synaptic complexes and align this new information with emerging cellular single-molecule microscopy studies as well as with previous studies of DNA-PK’s function in repair.

The Mediator complex is an essential coregulator of RNA polymerase II transcription. More recent developments suggest Mediator functions as a link between transcription regulation, genome organisation and DNA repair mechanisms including nucleotide excision repair, base excision repair, and homologous recombination. Dysfunctions of these processes are frequently associated with human pathologies, and growing evidence shows Mediator involvement in cancers, neurological, metabolic and infectious diseases. The detailed deciphering of molecular mechanisms of Mediator functions, using interdisciplinary approaches in different biological models and considering all functions of this complex, will contribute to our understanding of relevant human diseases.

Cellular and molecular responses to DNA damage are highly orchestrated and dynamic, acting to preserve the maintenance and integrity of the genome. Histone proteins bind DNA and organize the genome into chromatin. Post-translational modifications of histones have been shown to play an essential role in orchestrating the chromatin response to DNA damage by regulating the DNA damage response pathway. Among the histone modifications that contribute to this intricate network, histone ADP-ribosylation (ADPr) is emerging as a pivotal component of chromatin-based DNA damage response (DDR) pathways. In this review, we survey how histone ADPr is regulated to promote the DDR and how it impacts chromatin and other histone marks. Recent advancements have revealed histone ADPr effects on chromatin structure and the regulation of DNA repair factor recruitment to DNA lesions. Additionally, we highlight advancements in technology that have enabled the identification and functional validation of histone ADPr in cells and in response to DNA damage. Given the involvement of DNA damage and epigenetic regulation in human diseases including cancer, these findings have clinical implications for histone ADPr, which are also discussed. Overall, this review covers the involvement of histone ADPr in the DDR and highlights potential future investigations aimed at identifying mechanisms governed by histone ADPr that participate in the DDR, human diseases, and their treatments.

Eukaryotic DNA replication is a tightly controlled process that occurs in two main steps, i.e., licensing and firing, which take place in the G1 and S phases of the cell cycle, respectively. In Saccharomyces cerevisiae, the budding yeast, replication origins contain consensus sequences that are recognized and bound by the licensing factor Orc1–6, which then recruits the replicative Mcm2–7 helicase. By contrast, mammalian initiation sites lack such consensus sequences, and the mammalian ORC does not exhibit sequence specificity. Studies performed over the past decades have identified replication initiation sites in the mammalian genome using sequencing-based assays, raising the question of whether replication initiation occurs at confined sites or in broad zones across the genome. Although recent reports have shown that the licensed MCMs in mammalian cells are broadly distributed, suggesting that ORC-dependent licensing may not determine the initiation sites/zones, they are predominantly located upstream of actively transcribed genes. This review compares the mechanism of replication initiation in yeast and mammalian cells, summarizes the sequencing-based technologies used for the identification of initiation sites/zones, and proposes a possible mechanism of initiation-site/zone selection in mammalian cells. Future directions and challenges in this field are also discussed.

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.