剪切修复偶联因子1的结构与功能

剪切修复偶联因子1是一种重要的DNA修复因子,参与了包括核苷酸剪切修复和重组修复在内的多种DNA修复过程。本文主要介绍了该因子在DNA修复过程中的作用和机制,及在肿瘤治疗和衰老过程中的可能作用。     

DNA切除修复与转录偶联

细胞DNA受到某些环境理化因子损伤后,其中活性转录基因和DNA转录链上的损伤被优先切除修复,这种DNA选择性修复直接与基因转录过程偶联．在大肠杆菌中已分离到实现此功能的转录修复偶联因子（TRCF）,是由mdf基因编码的一种具有ATPase活性的DNA结合蛋白．在真核细胞中,发现某些DNA修复蛋白也在DNA转录中起作用,如人DNA切除修复基因ERCG－3编码产物,是转录因子TFⅡH中最大亚基p89,酵母切除修复基因RAD3就是编码因子b的最大亚基p85．     

北方地区汉族人核苷酸剪切修复基因mRNA的表达与头颈鳞癌发病风险的初步研究

目的:探讨我国北方汉族人核苷酸剪切修复(Nucleotide excision repair,NER)基因mRNA的表达水平与头颈鳞癌发病风险的相关性,为头颈鳞癌的诊断提供新的生物学标志物。方法:收集205例头颈鳞癌患者和176例健康对照,均为我国北方地区汉族人。通过实时定量聚合酶链反应实验检测研究对象外周血淋巴细胞中的5个核心核苷酸剪切修复基因mRNA的相对表达情况。对病例和对照之间一般特征的分布差异进行卡方检验,通过Wilcoxon秩和检验计算不同基因的mRNA表达水平的差异。采用logistic回归计算优势比(OR值)及95%置信区间(95%CI)。此外,通过ROC曲线评价NER基因模型的诊断价值。结果:病例组DDB1的mRNA表达低于对照组(P=0.075)。在logistic回归分析中,矫正年龄、性别、吸烟状况和饮酒因素后,DDB1的mRNA相对表达水平与SCCHN患病风险关系的ORs,在第二、第三和第四四分位数水平中分别为1.90(95%CI,1.02-3.54)、1.54(95%CI,0.82-2.87)和1.88(95%CI,1.00-3.52),分布与其mRNA的高表达水平相比。此外,DDB1(Ptrend=0.036)的蛋白表达水平降低与SCCHN风险增加之间也存在剂量反应关系)。ROC曲线提示DDB1表达水平与性别结合的效应模型中AUC显著改善(P=0.046)。结论:我国北方汉族DDB1的mRNA相对表达的降低与SCCHN患病风险的增加显著相关。     

西北地区汉族人核苷酸剪切修复蛋白的表达与头颈鳞癌发病风险的相关性研究

目的:初步探讨西北地区汉族人核苷酸剪切修复蛋白表达水平与头颈鳞癌发病风险的相关性,从翻译水平为头颈鳞癌提供新的筛检标志物。方法:收集118例头颈鳞癌患者和88例健康对照,均为西北地区汉族人。通过反向蛋白芯片实验检测研究对象外周血淋巴细胞中的5个核心核苷酸剪切修复蛋白的相对表达水平,采用卡方检验分析两组间一般特征的差异,并计算蛋白相对表达水平间的差异,logistic回归计算OR值及95%CI,最后通过绘制接受者操作特性曲线评价模型的诊断价值。结果:病例组XPB (Xeroderma pigmentosum, complementation group B)的表达水平显著低于对照组(P=0.013)。Logistic回归分析结果显示XPB高表达者相比,其低表达者头颈鳞癌患病风险的OR为1.74(95%CI,0.99-3.06)。此外,XPB的蛋白表达水平降低与SCCHN风险增加之间存在剂量反应关系(P_(trend)=0.042)。最后,我们通过接受者操作特性曲线计算曲线下面积,评估XPB表达水平的效应对于头颈鳞癌易感性筛检能力。包含XPB表达水平的效应模型中曲线下面积显著改善(P=0. 048)。结论:在西北地区汉族人中XPB的相对表达水平的降低与头颈鳞癌患病风险的增加相关。XPB表达水平的降低可能在既往吸烟者的头颈鳞癌患病风险中发挥更重要的作用。     

DNA修复基因RAD24的分子克隆和序列分析

利用缺口修复（ｇａｐｒｅｐａｉｒ）方法克隆啤酒酵母（Ｓ．ｃｅｒｅｖｉｓｉａｅ）野生型ＲＡＤ２４基因,并将其亚克隆到Ｍ１３ｍｐ１８和Ｍ１３ｍｐ１９,用双脱氧末端终止法对该基因的两条链均进行了序列测定,ＤＮＡＳｔｒｉｄｅｒ程序分析显示该基因编码２６８个氨基酸的蛋白质,基因缺失试验表明,该基因为细胞生存所必需。     

WT1基因在转录调控中的作用

ＷＴ１基因于１９９０年发现,在某些组织中特异表达,它的蛋白质产物在转录调控中起一种双向转录因子作用。它自身表达也受很多因素的调控。西方综述了近年来国内外关于ＷＴ１基因在转录调失方面的作用的研究进展。     

核苷酸切除修复对UVB光损伤的识别机制

中波紫外线（UVB）会对皮肤造成各种损伤,这些都根源于UVB对皮肤细胞DNA的光损伤。光损伤产物主要有环丁烷嘧啶二聚体（CPD）和64光产物（6-4PP）两类,还包括少量的氧化损伤。CPD和6-4PP的修复是由核苷酸切除修复（NER）执行的。NER可分为全基因组核苷酸切除修复（GGR）和转录耦联核苷酸切除修复（TCR）两个亚途径。识别因子XPC通过一种不直接识别损伤本身的机制在GGR识别过程中发挥作用;在TCR识别过程中强调了关键因子CSB单体及二聚体两种形式的转换。在染色质水平上,DDB介导的泛素化作用是NER识别过程中重要的调控要素。另外,完成使命的识别因子的最终走向也是NER途径中的一个重要环节。通过分析上述生化过程,较清楚地总结了GGR及TCR对UVB导致的光损伤的识别机制。     

转录调控因子Fox的功能及分子机制研究进展

Fox (Forkhead box)蛋白家族有19个亚族, 它们通过结合DNA, 激活或抑制目的基因的转录活性, 同时还能参与细胞信号转导、 细胞周期调控和新陈代谢的调节, 在生物体发育及其成熟的组织器官中均能发挥重要作用, 目前, 有关Fox蛋白家族的功能及分子机制已逐步成为免疫学、 遗传学、 医学以及肿瘤学领域的研究热点。本文综述了Fox家族成员的命名及分类、 蛋白结构及其DNA识别机制以及该家族成员如何参与Hh, TGF-β/SMAD, MAPK, Wnt/β-catenin和IGF信号通路的调控。Fox家族可调控线虫的咽、 果蝇的唾液腺以及哺乳动物的肝脏和眼睛等器官的发育, 能够影响细胞周期, 其家族成员FoxA可以和CREB、 GR结合调控新陈代谢。不同物种的Fox家族成员个数存在差异, 并且受到严格的进化选择。对其功能和分子进化机制进一步研究可为阐明生物的发育机理和人类疾病的防治提供新的思路。     

中介因子复合体在基因转录调控中的作用

近二十年来,人类在不断探索基因转录调控的机制方面取得了长足的进步,其中包括对中介因子复合体(mediator complex)的克隆、鉴定及作用机制的研究。中介因子是由20多种不同蛋白亚基组成的复合体,广泛存在于各种真核生物的细胞中,并且与RNA聚合酶一起构成RNA聚合酶Ⅱ全酶。中介因子复合体可与转录因子和RNA聚合酶Ⅱ相互作用,因而在基因转录过程中发挥着桥梁的作用。中介因子复合体不但能够促进基因转录的激活,有时也能抑制基因转录。本文总结了中介因子复合体的组成、结构及功能方面的研究进展。     

核苷酸切除修复基因XPA反义RNA表达载体的构建及其抑瘤功能

采用RT PCR方法扩增出 4 2 6bp着色性干皮病A(xerodermapigmentosumgroupA ,XPA)cDNA片段 (2～ 4 2 7bp) ,反向插入pcDNA3 1质粒构建XPA反义RNA表达载体 .经测序证实 ,该片段序列与XPAmRNA对应片段完全互补 .通过脂质体Lipofectamine 2 0 0 0将重组质粒转染肺癌A5 4 9细胞 ,RT PCR检测表明转染XPA反义RNA重组质粒能够抑制肺癌细胞XPAmRNA表达 ;MTT实验表明转染XPA反义RNA的肺癌细胞对顺铂敏感性增强 .本研究为深入探讨NER途径基因功能及临床克服肿瘤耐药提出了一个新的思路     

Efficient repair of cyclobutane pyrimidine dimers at mutational hot spots is restored in complemented Xeroderma pigmentosum group C and trichothiodystrophy/xeroderma pigmentosum group D cells

Xeroderma pigmentosum (XP) and trichothiodystrophy (TTD) are rare heritable diseases. Patients suffering from XP and 50% of TTD afflicted individuals are photosensitive and have a high susceptibility to develop skin tumors. One solution to alleviating symptoms of these diseases is to express the deficient cDNAs in patient cells as a form of gene therapy. XPC and TTD/XPD cell lines were complemented using retroviral transfer. Expressed wild-type XPC or XPD cDNAs in these cells restored the survival to UVC radiation to wild-type levels in the respective complementation groups. Although complemented XP cell lines have been studied for years, data on cyclobutane pyrimidine dimer (CPD) repair in these cells at different levels are sparse. We demonstrate that CPD repair is faster in the complemented lines at the global, gene, strand specific, and nucleotide specific levels than in the original lines. In both XPC and TTD/XPD complemented lines, CPD repair on the non-transcribed strand is faster than that for the MRC5SV line. However, global repair in the complemented cell lines and MRC5SV is still slower than in normal human fibroblasts. Despite the slower global repair rate, in the complemented XPC and TTD/XPD cells, almost all of the CPDs at "hotspots" for mutation in the P53 tumor database are repaired as rapidly as in normal human fibroblasts. Such evaluation of repair at nucleotide resolution in complemented nucleotide excision repair deficient cells presents a crucial way to determine the efficient re-establishment of function needed for successful gene therapy, even when full repair capacity is not restored.     

The Mechanism of Nucleotide Excision Repair-Mediated UV-Induced Mutagenesis in Nonproliferating Cells

Following the irradiation of nondividing yeast cells with ultraviolet (UV) light, most induced mutations are inherited by both daughter cells, indicating that complementary changes are introduced into both strands of duplex DNA prior to replication. Early analyses demonstrated that such two-strand mutations depend on functional nucleotide excision repair (NER), but the molecular mechanism of this unique type of mutagenesis has not been further explored. In the experiments reported here, an ade2 adeX colony-color system was used to examine the genetic control of UV-induced mutagenesis in nondividing cultures of Saccharomyces cerevisiae. We confirmed a strong suppression of two-strand mutagenesis in NER-deficient backgrounds and demonstrated that neither mismatch repair nor interstrand crosslink repair affects the production of these mutations. By contrast, proteins involved in the error-prone bypass of DNA damage (Rev3, Rev1, PCNA, Rad18, Pol32, and Rad5) and in the early steps of the DNA-damage checkpoint response (Rad17, Mec3, Ddc1, Mec1, and Rad9) were required for the production of two-strand mutations. There was no involvement, however, for the Pol η translesion synthesis DNA polymerase, the Mms2-Ubc13 postreplication repair complex, downstream DNA-damage checkpoint factors (Rad53, Chk1, and Dun1), or the Exo1 exonuclease. Our data support models in which UV-induced mutagenesis in nondividing cells occurs during the Pol ζ-dependent filling of lesion-containing, NER-generated gaps. The requirement for specific DNA-damage checkpoint proteins suggests roles in recruiting and/or activating factors required to fill such gaps.     

Focus: 50 Years of DNA Repair: The Yale Symposium Reports: The Awakening of DNA Repair at Yale

As a graduate student with Professor Richard Setlow at Yale in the late 1950s, I studied the effects of ultraviolet and visible light on the syntheses of DNA, RNA, and protein in bacteria. I reflect upon my research in the Yale Biophysics Department, my subsequent postdoctoral experiences, and the eventual analyses in the laboratories of Setlow, Paul Howard-Flanders, and myself that constituted the discovery of the ubiquitous pathway of DNA excision repair in the early 1960s. I then offer a brief perspective on a few more recent developments in the burgeoning DNA repair field and their relationships to human disease.     

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

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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.     

Focus: 50 Years of DNA Repair: The Yale Symposium Reports: Early Days of DNA Repair: Discovery of Nucleotide Excision Repair and Homology-Dependent Recombinational Repair

The discovery of nucleotide excision repair in 1964 showed that DNA could berepaired by a mechanism that removed the damaged section of a strand andreplaced it accurately by using the remaining intact strand as the template.This result showed that DNA could be actively metabolized in a process that hadno precedent. In 1968, experiments describing postreplication repair, a processdependent on homologous recombination, were reported. The authors of thesepapers were either at Yale University or had prior Yale connections. Here werecount some of the events leading to these discoveries and consider the impacton further research at Yale and elsewhere.