Role of HMGA1 proteins in DNA damage and excision repair

HMGA1 蛋白在 DNA 损伤和切除修复中的作用

• 批准号: 6926730
• 负责人: RAYMOND REEVES
• 金额: $ 28.18万
• 依托单位: WASHINGTON STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-04-01 至 2009-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6926730
• 关键词: DNA binding protein; DNA damage; DNA repair; Xenopus oocyte; gene complementation; gene induction /repression; genetic transcription; protein structure function; pyrimidine dimers; transcription factor; ultraviolet radiation

DESCRIPTION (provided by applicant): Mammalian HMGA1 chromatin proteins are architectural transcription factors that specifically bind to the minor groove of AT-rich DNA regions, thus making them prime candidates as participants in the recognition and repair of UV-induced photo-lesions (e.g., cis-syn cyclobutane pyrimidine dinners, or CPDs). Indeed, over-expression of HMGA1 proteins inhibits the repair of CPDs both in vivo and in vitro and also significantly increases the sensitivity of cells to UV-induced killing, a trait that is characteristic of excision repair deficient cells. We hypothesize that HMGA1 proteins inhibit both global-genomic and gene-specific nucleotide and base excision repair (i.e., NER and BER) in at least two ways: (a) by physically influencing the processes of both lesion formation and removal in DNA and chromatin substrates; and, (b) by transcriptional repression of specific NER and BER repair genes. The Specific Aims of the research are to: (1) Determine the efficiency of both global-genomic and gene-specific NER and BER of DNA lesions, such as CPDs and alkylated bases, in cells in which the intracellular concentrations of HMGA1 proteins can be experimentally regulated as well as in cells with naturally occurring differences in HMGA1 levels; (2) Employ genetic complementation and other in vivo approaches to distinguish between the effects of HMGA1 over-expression on repression of excision repair gene transcription and inhibition of other NER and BER processes; (3) Measure the effects of DNA lesions, such as CPDs or uracil, at specific sites in synthetic DNA substrates on HMGA1 binding to its cognate DNA sequences before and after nucleosome assembly in vitro; and, (4) Measure the effects of bound HMGA1 proteins on NER or BER of its cognate DNA binding sequences containing site-specific lesions before and after nucleosome assembly using either Xenopus oocyte nuclear repair extracts (NER) or purified human proteins (BER). The HMGA genes are the only known oncogenes that code for bona fide chromosome structural proteins and whose over-expression is considered a diagnostic feature of many human cancers. Thus, elucidation of the mechanisms by which the HMGA proteins inhibit repair of DNA lesions will provide important new insights into the underlying causes of the accumulation of genetic mutations, and the occurrence of genomic instabilities, that are the hallmarks of cancerous cells. These studies could also lead to the identification of new molecular targets for therapeutic treatment of a number of human malignancies.

描述（由申请人提供）：哺乳动物HMGA1染色质蛋白是建筑转录因子，特异性与富裕的DNA区域的小凹槽相结合，从而使其成为识别和修复紫外线诱导的照片的参与者的主要候选者（例如，Cis-syn cyclobutane pyclobutane pylimidine pyrimidine pyrimidine dinners或cpds）。 实际上，HMGA1蛋白的过表达抑制了体内和体外CPD的修复，并且还显着提高了细胞对紫外线诱导的杀伤的敏感性，紫外线诱导的杀伤是切除修复缺陷细胞的特征的特征。 我们假设HMGA1蛋白抑制了全球基因组和基因特异性核苷酸和碱基切除修复（即NER和BER）至少两种方式：（a）通过物理影响​​DNA和染色质sibstrates中病变形成和去除的过程； （b）通过转录抑制特定的NER和BER修复基因。 该研究的具体目的是：（1）确定全球基因组和基因特异性NER和DNA病变的BER的效率，例如CPD和烷基化碱，在HMGA1蛋白质的细胞内浓度可以通过实验调节HMGA1蛋白的细胞内浓度，并且在HMGA1水平差异的天然差异都可以受到实验调节； （2）采用遗传互补和其他体内方法来区分HMGA1过表达对抑制切除修复基因转录的影响以及对其他NER和BER过程的抑制； （3）测量合成DNA底物中特定位点的DNA病变（例如CPD或尿嘧啶）在体外结合其与其同源DNA序列的HMGA1中的特定位点的作用； （4）使用Xenopus卵母细胞核修复提取物（NER）或纯化的人蛋白（BER）来测量结合HMGA1蛋白对含有位点特异性病变的同源DNA结合序列的NER或BER的影响。 HMGA基因是唯一已知的肿瘤基因，用于编码真正的染色体结构蛋白，其过表达被认为是许多人类癌症的诊断特征。 因此，阐明HMGA蛋白抑制DNA病变修复的机制将为遗传突变积累的根本原因提供重要的新见解，以及基因组不稳定性的发生，即癌细胞的标志。 这些研究还可能导致鉴定出新的分子靶标，以治疗许多人类恶性肿瘤。