Involvement of Nucleotide Excision and Mismatch Repair Mechanisms in Double Strand Break Repair

Living organisms are constantly threatened by environmental DNA-damaging agents, including UV and ionizing radiation (IR). Repair of various forms of DNA damage caused by IR is normally thought to follow lesion-specific repair pathways with distinct enzymatic machinery. DNA double strand break is one of the most serious kinds of damage induced by IR, which is repaired through double strand break (DSB) repair mechanisms, including homologous recombination (HR) and non-homologous end joining (NHEJ). However, recent studies have presented increasing evidence that various DNA repair pathways are not separated, but well interlinked. It has been suggested that non-DSB repair mechanisms, such as Nucleotide Excision Repair (NER), Mismatch Repair (MMR) and cell cycle regulation, are highly involved in DSB repairs. These findings revealed previously unrecognized roles of various non-DSB repair genes and indicated that a successful DSB repair requires both DSB repair mechanisms and non-DSB repair systems. One of our recent studies found that suppressed expression of non-DSB repair genes, such as XPA, RPA and MLH1, influenced the yield of IR induced micronuclei formation and/or chromosome aberrations, suggesting that these genes are highly involved in DSB repair and DSB-related cell cycle arrest, which reveals new roles for these gene products in the DNA repair network. In this review, we summarize current progress on the function of non-DSB repair-related proteins, especially those that participate in NER and MMR pathways, and their influence on DSB repair. In addition, we present our developing view that the DSB repair mechanisms are more complex and are regulated by not only the well known HR/NHEJ pathways, but also a systematically coordinated cellular network.Key Words: Ionizing radiation (IR), DNA damage, DSB repair, NER, MMR and cell cycle.     

DNA错配修复、染色体不稳定和肿瘤的关系

DNA错配修复系统可以识别并纠正DNA复制过程中出现的错误.保证基因组的稳定性和完整性.错配修复系统缺陷可能导致遗传物质发生突变,引发恶性肿瘤.肿瘤患者经常表现出染色体不稳定,具体表现为微卫星不稳定性和杂合性缺失.本文就DNA错配修复、染色体不稳定和肿瘤之间的联系予以综述.     

Nucleotide Excision Repair in the Third Kingdom

Nucleotide excision repair, a general repair mechanism for removing DNA damage, is initiated by dual incisions bracketing the lesion. In procaryotes, the dual incisions result in excision of the damage in 12- to 13-nucleotide-long oligomers, and in eucaryotes they result in excision of the damage in the form of 24- to 32-nucleotide-long oligomers. We wished to find out if Archaea perform excision repair. Using cell extracts from Methanobacterium thermoautotrophicum, we found that this organism removes UV-induced (6-4) photoproducts in the form of 10- to 11-mers by incising the sixth to seventh phosphodiester bond 5′ to the damage and the fourth phosphodiester bond 3′ to the damage.     

Interactome of Base and Nucleotide Excision DNA Repair Systems

Molecular Biology - The base and nucleotide excision DNA repair (BER and NER) systems are aimed at removing specific types of damaged DNA, i.e., oxidized, alkylated, or deaminated bases in the case...     

Nucleotide Excision Repair Disorders and the Balance Between Cancer and Aging

Cancer incidence increases with age and is driven by accumulation of mutations in the DNA. In many so-called premature aging disorders, cancer appears earlier and at elevated rates. These diseases are predominantly caused by genome instability and present with symptoms, including cancer, resembling “segments” of aging and are thus often referred to as “segmental progerias”. Two related segmental progerias, Cockayne syndrome (CS) and trichothiodystrophy (TTD), don’t fit this pattern. Although caused by defects in genome maintenance via the nucleotide excision DNA repair (NER) pathway and displaying severe progeroid symptoms, CS and TTD patients appear to lack any cancer predisposition. More strikingly, genetic defects in the same NER pathway, and in some cases even within the same gene, XPD, can also give rise to disorders with greatly elevated cancer rates but without progeria (xeroderma pigmentosum). In this review, we will discuss the connection between genome maintenance, aging and cancer in light of a new mouse model of XPD disease.     

Regulation of Nucleotide Excision Repair by Nuclear Lamin B1

The nuclear lamins play important roles in the structural organization and function of the metazoan cell nucleus. Recent studies on B-type lamins identified a requirement for lamin B1 (LB1) in the regulation of cell proliferation in normal diploid cells. In order to further investigate the function of LB1 in proliferation, we disrupted its normal expression in U-2 OS human osteosarcoma and other tumor cell lines. Silencing LB1 expression induced G1 cell cycle arrest without significant apoptosis. The arrested cells are unable to mount a timely and effective response to DNA damage induced by UV irradiation. Several proteins involved in the detection and repair of UV damage by the nucleotide excision repair (NER) pathway are down-regulated in LB1 silenced cells including DDB1, CSB and PCNA. We propose that LB1 regulates the DNA damage response to UV irradiation by modulating the expression of specific genes and activating persistent DNA damage signaling. Our findings are relevant to understanding the relationship between the loss of LB1 expression, DNA damage signaling, and replicative senescence.     

Enzymes of Direct,Excision, and Mismatch DNA Repair in Pro- and Eukaryotes and Their Biological Role

DNA repair is of paramount importance to protect the integrity of the cell genome. Over the last decade, DNA repair enzymes have been intensely studied owing to their role in mutagenesis, carcinogenesis, and aging. This led to discoveries of novel enzymes, protein factors as well as their intermolecular complexes involved in the repair process. Alternative pathways of cell response to the same type of damage were identified. Data on the tertiary structure of a number of enzymes and mechanisms of their molecular interaction were obtained. This paper is an overview of latest advances in the research of cell response to DNA damage via direct, base and nucleotide excision, and mismatch DNA repair.     

Analysis of Ribonucleotide Removal from DNA by Human Nucleotide Excision Repair

Ribonucleotides are incorporated into the genome during DNA replication. The enzyme RNase H2 plays a critical role in targeting the removal of these ribonucleotides from DNA, and defects in RNase H2 activity are associated with both genomic instability and the human autoimmune/inflammatory disorder Aicardi-Goutières syndrome. Whether additional general DNA repair mechanisms contribute to ribonucleotide removal from DNA in human cells is not known. Because of its ability to act on a wide variety of substrates, we examined a potential role for canonical nucleotide excision repair in the removal of ribonucleotides from DNA. However, using highly sensitive dual incision/excision assays, we find that ribonucleotides are not efficiently targeted by the human nucleotide excision repair system in vitro or in cultured human cells. These results suggest that nucleotide excision repair is unlikely to play a major role in the cellular response to ribonucleotide incorporation in genomic DNA in human cells.     

Cell Death is Associated with Reduced Base Excision Repair During Chronic Alcohol Administration in Adult Rat Brain

The cell death cascades in different brain regions namely hippocampus and frontal cortex of rats fed with 10% (v/v) ethanol for 12 weeks, was examined. After Western blotting, different cell death associated proteins displayed differential activation in the two regions observed. In hippocampus, activated caspase-3 and caspase-7 resulted in subsequent cleavage of poly(ADP-ribose) polymerase-1 (PARP-1). Cytochrome c release to cytosol and apoptosis inducing factor (AIF) translocation to nucleus was marginal. B-cell leukemia/lymphoma-2 (Bcl-2) translocation to cytosol was significant whereas Bcl-2-associated X protein (Bax) and Bcl-associated death protein (Bad) were largely located in cytosol. Further, upregulation of N-methyl d-aspartate receptor subunit 1 (NMDAR1), N-methyl d-aspartate receptor subunit 2B (NMDAR2B), N-methyl d-aspartate receptor subunit 2C (NMDAR2C) and activation of calpains were observed. In frontal cortex, caspase-3 activation, cleavage of PARP-1 and nuclear translocation of AIF were more pronounced. Moreover, cytochrome c release to cytosol, Bcl-2 translocation to cytosol was evident. However, levels of Bax, Bad, NMDA receptor subunits, and calpains were unaffected. Apoptosis was further substantiated by in situ staining for terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL). Results of the current study revealed that frontal cortex exhibits a higher level of ethanol-induced apoptosis relative to hippocampus. DNA polymerase beta assay and immunoblot showed significant loss in base excision repair in ethanol treated group.     

人XPB基因在核苷酸剪切修复和基因转录中的分子机制

核苷酸剪切修复(NER)途径是维持生物体基因组稳定的重要机制。人着色性干皮病B组(xeroderma pigmentosum group B,XPB)基因又名ERCC3基因,它既是NER途径不可缺少的成员又是转录因子TFIIH的最大p89亚基。它是具有从3’端→5’端依赖ATP的单链DNA解旋酶活性的蛋白质,执行依赖DNA的ATP酶和解旋酶功能,在损伤DNA修复和基因转录中均起重要作用,并将两者有机偶联。该基因突变将导致3种不同的遗传疾病：着色性干皮病(xeroderma pigmentosum,XP),科凯恩氏综合征(cockayne’s syndrome,CS),毛发硫营养不艮(trichothiodystrophy,TTD)。其基因型通过在DNA修复和转录中的功能与表型联系起来。另外,XPB与p53存在物理和功能上的相互作用。现从XPB的3个方面即“一个基因,两种功能,3种疾病”作一综述。     

Conformational Dynamics of Human AP Endonuclease in Base Excision and Nucleotide Incision Repair Pathways

Abstract

APE1 is a multifunctional enzyme that plays a central role in base excision repair (BER) of DNA. APE1 is also involved in the alternative nucleotide incision repair (NIR) pathway. We present an analysis of conformational dynamics and kinetic mechanisms of the full-length APE1 and truncated NΔA61-APE1 lacking the N-terminal 61 amino acids (REF1 domain) in BER and NIR pathways. The action of both enzyme forms were described by identical kinetic schemes, containing four stages corresponding to formation of the initial enzyme-substrate complex and isomerization of this complex; when a damaged substrate was present, these stages were followed by an irreversible catalytic stage resulting in the formation of the enzyme-product complex and the equilibrium stage of product release. For the first time we showed, that upon binding AP-containing DNA, the APE1 structure underwent conformational changes before the chemical cleavage step. Under BER conditions, the REF1 domain of APE1 influenced the stability of both the enzyme-substrate and enzyme-product complexes, as well as the isomerization rate, but did not affect the rates of initial complex formation or catalysis. Under NIR conditions, the REF1 domain affected both the rate of formation and the stability of the initial complex. In comparison with the full-length protein, NΔA61-APE1 did not display a decrease in NIR activity with a dihydrouracil-containing substrate. BER conditions decrease the rate of catalysis and strongly inhibit the rate of isomerization step for the NIR substrates. Under NIR conditions AP-endonuclease activity is still very efficient.     

DNA ADP-Ribosylation Stalls Replication and Is Reversed by RecF-Mediated Homologous Recombination and Nucleotide Excision Repair

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