切除修复基因

切除修复是生物体中一种重要的修复ＤＮＡ损伤的方式，它是生物体对周围物化因素攻击产生耐受性的基础，保证了遗传信息的准确传递和生命活动的正常进行。已发现在原核和真核细胞中都存在切除修复。本文综述有关大肠杆菌、酿酒酵母、鼠和人细胞切除修复基因研究的进展。     

DNA损伤修复基本方式的研究进展

DNA损伤修复基因可修复由不同原因导致的DNA损伤 ,从而保护遗传信息的完整性。DNA损伤修复有 3种基本形式 ,即碱基切除修复、核苷酸切除修复和错配修复。本文综述了DNA损伤修复 3种基本形式的研究进展情况并讨论了DNA链断裂重组和重接合修复及DNA聚合酶绕道修复DNA损伤     

DNA修复基因：核苷酸切除修复基因单核苷酸多态与癌症发生风险的研究进展

单核苷酸多态(single nucleotide polymorphism，SNP)是目前受到高度重视的全新一代遗传标记，将是今后基因组学研究的一大主要工具。DNA修复系统基因在维持基因组功能整体性，修复致癌因素所致的损伤及抗癌过程中有着重要作用。一些普通的及新的DNA修复基因单核苷酸多态与癌症发生风险的关联已经被验证或正在被验证，本文仅就核苷酸切除修复基因单核苷酸多态与癌症发生风险的研究进展作一综述。     

DNA修复与顺铂耐药

顺铂的主要药理机制是损伤细胞DNA，肿瘤细胞DNA修复能力增强则会导致其对顺铂耐药。目前研究发现，DNA修复途径中的核苷酸切除修复、错配修复、碱基切除修复均与顺铂耐药有关，核苷酸切除修复为其中最为重要的途径。     

6A8α-甘露糖苷酶表达抑制使Jurkat细胞间的黏附性增强

目的　探讨 6A8α 甘露糖苷酶表达抑制对人T淋巴母细胞Jurkat生物学行为的影响。方法　采用反义核酸技术抑制细胞中 6A8α 甘露糖苷酶的表达。用PCR检测转导基因在基因组中的整合。用单抗 6A8作探针 ,Western印迹和免疫荧光染色流式细胞术检测细胞中 6A8α 甘露糖苷酶的表达状况。用ConA结合试验检测细胞蛋白质的糖基化改变。普通光学显微镜观察细胞的形态。用DNA芯片技术分析AS细胞和M细胞mRNA表达的差异。用RT PCR和免疫荧光染色流式细胞术验证基因芯片结果的可靠性。结果　制备了 6A8α 甘露糖苷酶表达抑制的Jurkat细胞 (AS)。与对照细胞 (野生型细胞W和转导空载体的细胞M)相比 ,AS细胞在培养中黏附成大团块。DNA芯片分析见与M细胞相比 ,AS细胞中有 19个基因的表达上调 ,2 0个基因的表达下调。上调的基因中有一些与细胞间黏附相关 ,如CD5 4、CD11a、CD2 4、CD82、整合素αX、整合素α7、整合素αE、IL 1R、IL 2Rγ和TNFSF9。RT PCR肯定了DNA芯片分析的可靠性。免疫荧光染色流式细胞术进一步肯定了CD5 4和CD11a在AS细胞表达的上调。结论　 6A8α 甘露糖苷酶表达抑制使Jurkat细胞间黏附增强 ,CD5 4和CD11a等黏附分子表达的增高可能与此相关。     

发现DNA修复新机制

据《自然》2010年10月3日报道（Nature,10—3—2010）,美国范德比特大学、宾夕法尼亚州立大学及匹兹堡大学的研究人员发现了一种DNA修复酶检测并修复遗传密码碱基损伤的新机制。通常认为双螺旋结构的DNA是非常稳定的,但事实上DNA性质高度活跃。由于人体细胞内正常的化学活动及自然环境放射和毒性物质的作用,人体的每个细胞每天约有一百万个DNA碱基发生损伤。     

6A8α-甘露糖甘酶表达抑制使Jurkat细胞间的黏附性增强

目的 探讨6A8α-甘露糖苷酶表达抑制对人T淋巴母细胞Judua生物学行为的影响。方法 采用反义核酸技术抑制细胞中6A8α-甘露糖苷酶的表达。用PCR检测转导基因在基因组中的整合。用单抗6A8作探针，Westem印迹和免疫荧光染色流式细胞术检测细胞中6A8α-甘露糖苷酶的表达状况。用Con A结合试验检测细胞蛋白质的糖基化改变。普通光学显微镜观察细胞的形态。用DNA芯片技术分析AS细胞和M细胞mRNA表达的差异。用RT-PCR和免疫荧光染色流式细胞术验证基因芯片结果的可靠性。结果 制备了6A8α-甘露糖苷酶表达抑制的Jurkat细胞(AS)。与对照细胞(野生型细胞W和转导空载体的细胞M)相比，AS细胞在培养中黏附成大团块。DNA芯片分析见与M细胞相比，AS细胞中有19个基因的表达上调，加个基因的表达下调。上调的基因中有一些与细胞间黏附相关，如CD54、CD11a、CD24、CD82、整合紊αX、整合紊α7、整合素αE、IL-1R、IL-2Rγ和TNFSF9。RT-PCR肯定了DNA芯片分析的可靠性。免疫荧光染色流式细胞术进一步肯定了CD54和CD11a在AS细胞表达的上调。结论 6A8α-甘露糖苷酶表达抑制使Judua细胞间黏附增强，CD54和CD11a等黏附分子表达的增高可能与此相关。     

8-羟基脱氧鸟苷在乳腺癌中的表达及意义

目的 探讨8-羟基脱氧鸟苷(8-OHdG)在乳腺癌中的表达及意义.方法 ELISA法检测173例乳腺癌患者术前血清中8-OHdG表达水平;免疫组化法检测150例乳腺癌患者癌组织中8-OHdG的表达.结果 血清中8-OHdG的表达水平与癌组织中8-OHdG的表达存在相关性(P ＜0.05,r =0.163).多因素分析结果显示8-OHdG免疫组化染色阴性患者生存率更低(P＜0.01),而对导管内癌(n =140)患者更显著(P ＜0.001);血清中8-OHdG低表达与淋巴管及淋巴结转移显著相关.结论 血清中8-OHdG低表达及癌组织中8-OHdG低表达均与乳腺癌的浸润相关.8-OHdG染色阴性可能是乳腺癌患者死亡的一个独立预后因子.     

烷化剂诱导的哺乳动物修复相关基因表达

烷化剂可通过对DNA的直接攻击及使细胞处于应激状态而引起一系列反应。它可诱导许多基因表达的改变,参与细胞应激的多种功能,引起细胞对外界损伤剂的耐受,也参与癌变、突变的发生,其中诱导表达与修复相关基因可参与烷化修复的各个环节,提供细胞对烷化剂的抗性,与化学物致癌的预防相关。     

Essential role of β‐human 8‐oxoguanine DNA glycosylase 1 in mitochondrial oxidative DNA repair

8‐Oxoguanine (8‐OG) is the major mutagenic base lesion in DNA caused by reactive oxygen species (ROS) and accumulates in both nuclear and mitochondrial DNA (mtDNA). In humans, 8‐OG is primarily removed by human 8‐OG DNA glycosylase 1 (hOGG1) through the base excision repair (BER) pathway. There are two major hOGG1 isoforms, designated α‐ and β‐hOGG1, generated by alternative splicing, and they have distinct subcellular localization: cell nuclei and mitochondria, respectively. Using yeast two‐hybrid screening assays, we found that β‐ but not α‐hOGG1 directly interacts with the mitochondrial protein NADH:ubiquinone oxidoreductase 1 beta subcomplex 10 (NDUFB10), an integral factor in Complex 1 on the mitochondrial inner membrane. Using coimmunoprecipitation and immunofluorescence studies, we found that this interaction was greatly increased by hydrogen peroxide‐induced oxidative stress, suggesting that β‐ but not α‐hOGG1 is localized in the mitochondrial inner membrane. Analyses of nuclear and mtDNA damage showed that the β‐ but not α‐ hogg1 knockdown (KD) cells were severely defective in mitochondrial BER, indicating an essential requirement of β‐hOGG1 for mtDNA repair. β‐hogg1 KD cells were also found to be mildly deficient in Complex I activity, suggesting that β‐hOGG1 is an accessory factor for the mitochondrial integral function for ATP synthesis. In summary, our findings define β‐hOGG1 as an important factor for mitochondrial BER and as an accessory factor in the mitochondrial Complex I function. Mol. Mutagen. 2013. © 2012 Wiley Periodicals, Inc.     

Drosophila S3 ribosomal protein accelerates repair of 8-oxoguanine performed by human and mouse cell extracts

The S3 ribosomal protein of Drosophila melanogaster possesses various DNA repair activities, including the capacity to incise at apurinic/apyrimidinic (AP) sites and 8-oxo-7,8-dihydroguanine (8-oxoG) residues. We have recently hypothesized that this multifunctional protein may improve the efficiency of DNA base excision repair (BER) in mammalian cells. We have investigated the effect of pure GST-tagged Drosophila S3 on BER of different endogenous lesions performed by human and mouse cell extracts. Drosophila S3 significantly accelerated the BER of 8-oxoG (initiated by the bifunctional glycosylase OGG1). The stimulating effect was linked to the capacity of S3 to remove the 8-oxoG lesion and cleave the resulting AP site, rather than acceleration of downstream steps of the BER pathway (e.g., removal of 3' blocking fragments). No stimulating effect was observed on the BER of uracil, natural AP sites, and beta-lyase-cleaved AP sites. Heterologous expression of Drosophila S3 may be used to enhance 8-oxoG repair in human cells.     

Adenine DNA glycosylase activity of 14 Human MutY homolog (MUTYH) variant proteins found in patients with colorectal polyposis and cancer

Biallelic inactivating germline mutations in the base excision repair MUTYH (MYH) gene have been shown to predispose to MUTYH‐associated polyposis (MAP), which is characterized by multiple colorectal adenomas and carcinomas. In this study, we successfully prepared highly homogeneous human MUTYH type 2 recombinant proteins and compared the DNA glycosylase activity of the wild‐type protein and fourteen variant‐type proteins on adenine mispaired with 8‐hydroxyguanine, an oxidized form of guanine. The adenine DNA glycosylase activity of the p.I195V protein, p.G368D protein, p.M255V protein, and p.Y151C protein was 66.9%, 15.2%, 10.7%, and 4.5%, respectively, of that of the wild‐type protein, and the glycosylase activity of the p.R154H, p.L360P, p.P377L, p.452delE, p.R69X, and p.Q310X proteins as well as of the p.D208N negative control form was extremely severely impaired. The glycosylase activity of the p.V47E, p.R281C, p.A345V, and p.S487F proteins, on the other hand, was almost the same as that of the wild‐type protein. These results should be of great value in accurately diagnosing MAP and in fully understanding the mechanism by which MUTYH repairs DNA in which adenine is mispaired with 8‐hydroxyguanine. © 2010 Wiley‐Liss, Inc.     

Different organization of base excision repair of uracil in DNA in nuclei and mitochondria and selective upregulation of mitochondrial uracil-DNA glycosylase after oxidative stress

Oxidative stress in the brain may cause neuro-degeneration, possibly due to DNA damage. Oxidative base lesions in DNA are mainly repaired by base excision repair (BER). The DNA glycosylases Nei-like DNA glycosylase 1 (NEIL1), Nei-like DNA glycosylase 2 (NEIL2), mitochondrial uracil-DNA glycosylase 1 (UNG1), nuclear uracil-DNA glycosylase 2 (UNG2) and endonuclease III-like 1 protein (NTH1) collectively remove most oxidized pyrimidines, while 8-oxoguanine-DNA glycosylase 1 (OGG1) removes oxidized purines. Although uracil is the main substrate of uracil-DNA glycosylases UNG1 and UNG2, these proteins also remove the oxidized cytosine derivatives isodialuric acid, alloxan and 5-hydroxyuracil. UNG1 and UNG2 have identical catalytic domain, but different N-terminal regions required for subcellular sorting. We demonstrate that mRNA for UNG1, but not UNG2, is increased after hydrogen peroxide, indicating regulatory effects of oxidative stress on mitochondrial BER. To examine the overall organization of uracil-BER in nuclei and mitochondria, we constructed cell lines expressing EYFP (enhanced yellow fluorescent protein) fused to UNG1 or UNG2. These were used to investigate the possible presence of multi-protein BER complexes in nuclei and mitochondria. Extracts from nuclei and mitochondria were both proficient in complete uracil-BER in vitro. BER assays with immunoprecipitates demonstrated that UNG2-EYFP, but not UNG1-EYFP, formed complexes that carried out complete BER. Although apurinic/apyrimidinic site endonuclease 1 (APE1) is highly enriched in nuclei relative to mitochondria, it was apparently the major AP-endonuclease required for BER in both organelles. APE2 is enriched in mitochondria, but its possible role in BER remains uncertain. These results demonstrate that nuclear and mitochondrial BER processes are differently organized. Furthermore, the upregulation of mRNA for mitochondrial UNG1 after oxidative stress indicates that it may have an important role in repair of oxidized pyrimidines.     

A role for Myh1 in DNA repair after treatment with strand‐breaking and crosslinking chemotherapeutic agents

The highly conserved DNA glycosylase MutY is implicated in repair of oxidative DNA damage, in particular in removing adenines misincorporated opposite 7,8‐dihydro‐8‐oxoguanine (8‐oxo‐G). The MutY homologues (MutYH) physically associate with proteins implicated in replication, DNA repair, and checkpoint signaling, specifically with the DNA damage sensor complex 9‐1‐1 proteins. Here, we ask whether MutYH could have a broader function in sensing and repairing different types of DNA damage induced by conventional chemotherapeutics. Thus, we examined if deletion of the Schizosaccharomyces pombe MutY homologue, Myh1, alone or in combination with deletion of either component of the 9‐1‐1 sensor complex, influences survival after exposure to different classes of DNA damaging chemotherapeutics that do not act primarily by causing 8‐oxoG lesions. We show that Myh1 contributes to survival on genotoxic stresses induced by the oxidizing, DNA double strand break‐inducing, bleomycins, or the DNA crosslinking platinum compounds, particularly in a rad1 mutant background. Exposure of cells to cisplatin leads to a moderate overall accumulation of Myh1 protein. Interestingly, we found that DNA damage induced by phleomycin results in increased chromatin association of Myh1. Further, we demonstrate that Myh1 relocalizes to the nucleus after exposure to hydrogen peroxide or chemotherapeutics, most prominently seen after phleomycin treatment. These observations indicate a wider role of Myh1 in DNA repair and DNA damage‐induced checkpoint activation than previously thought. Environ. Mol. Mutagen. 54:327–337, 2013. © 2013 Wiley Periodicals, Inc.     

Requirement of the Saccharomyces cerevisiae APN1 gene for the repair of mitochondrial DNA alkylation damage

The Saccharomyces cerevisiae APN1 gene that participates in base excision repair has been localized both in the nucleus and the mitochondria. APN1 deficient cells (apn1Δ) show increased mutation frequencies in mitochondrial DNA (mtDNA) suggesting that APN1 is also important for mtDNA stability. To understand APN1‐dependent mtDNA repair processes we studied the formation and repair of mtDNA lesions in cells exposed to methyl methanesulfonate (MMS). We show that MMS induces mtDNA damage in a dose‐dependent fashion and that deletion of the APN1 gene enhances the susceptibility of mtDNA to MMS. Repair kinetic experiments demonstrate that in wild‐type cells (WT) it takes 4 hr to repair the damage induced by 0.1% MMS, whereas in the apn1Δ strain there is a lag in mtDNA repair that results in significant differences in the repair capacity between the two yeast strains. Analysis of lesions in nuclear DNA (nDNA) after treatment with 0.1% MMS shows a significant difference in the amount of nDNA lesions between WT and apn1Δ cells. Interestingly, comparisons between nDNA and mtDNA damage show that nDNA is more sensitive to the effects of MMS treatment. However, both strains are able to repair the nDNA lesions, contrary to mtDNA repair, which is compromised in the apn1Δ mutant strain. Therefore, although nDNA is more sensitive than mtDNA to the effects of MMS, deletion of APN1 has a stronger phenotype in mtDNA repair than in nDNA. These results highlight the prominent role of APN1 in the repair of environmentally induced mtDNA damage. Environ. Mol. Mutagen., 2009. © 2009 Wiley‐Liss, Inc.     

Deregulated expression of DNA polymerase β is involved in the progression of genomic instability

Deregulated expression of DNA polymerase beta (pol β) has been implicated in genomic instability that leads to tumorigenesis, yet the mechanisms underlying the pol β‐mediated genetic instability remain elusive. In this study, we investigated the roles of deregulated expression of pol β in spontaneous and xenobiotic‐induced genetic instability using mouse embryonic fibroblasts (MEFs) that express distinct pol β levels (wild‐type, null, and overexpression) as a model system. Three genetic instability endpoints, DNA strand breaks, chromosome breakage, and gene mutation, were examined under various expression levels of pol β by comet assay, micronuclei test, and hprt mutation assay. Our results demonstrate that neither pol β deficiency nor pol β overexpression is sufficient for accumulation of spontaneous DNA damage that promotes a hyperproliferation phenotype. However, pol β null cells exhibit increased sensitivity to exogenous DNA damaging agents with increased genomic instability compared with pol β wild‐type and overexpression cells. This finding suggests that a pol β deficiency may underlie genomic instability induced by exogenous DNA damaging agents. Interestingly, pol β overexpression cells exhibit less chromosomal or DNA damage, but display a higher hprt mutation frequency upon methyl methanesulfonate exposure compared with the other two cell types. Our results therefore indicate that an excessive amount of pol β may promote genomic instability, presumably through an error‐prone repair response, although it enhances overall BER capacity for induced DNA damage. Environ. Mol. Mutagen. 2012. © 2012Wiley Periodicals, Inc.     

The 8-hydroxydeoxyguanosine concentrations according to hormone therapy and S326C polymorphism of OGG1 gene in postmenopausal women

Background

The 8-hydroxydeoxyguanosine (8-OHdG) is widely used for determination of DNA damage since it is excised from oxidative damaged DNA with endonuclease repair enzymes coded by 8-oxoguanine DNA N-glycosylase gene (OGG1). The present study aimed at investigating whether hormone therapy (HT) may influence on the blood/urinary 8-OHdG levels and whether the level of 8-OHdG is different according to OGG1 S326C polymorphism in postmenopausal women receiving HT.

Methods

In 102 postmenopausal women receiving HT, the 8-OHdG levels were measured in the blood and urine using high performance liquid chromatography (HPLC) before HT and 3 months after HT. The genotyping of the S326C polymorphism of the OGG1 was performed by polymerase chain reaction (PCR) and restriction enzyme fragment length polymorphism (RFLP) analysis.

Results

After HT, mean blood 8-OHdG level significantly decreased compared to those before HT (P = 0.003), while urinary 8-OHdG level did not show any difference according to HT. Pre-HT level of 8-OHdG was not different according to OGG1 genotypes and similar finding was demonstrated in post-HT 8-OHdG concentration.

Conclusions

These findings imply that hormone therapy can reduce blood 8-OHdG concentration, one of the markers of oxidative damage. Further study is needed to confirm this association in larger population.