Role of HMGA1 proteins in DNA damage and excision repair

描述（由申请人提供）：哺乳动物HMGA1染色质蛋白是一种结构转录因子，专门结合到富含at的DNA区域的小凹槽上，因此使其成为识别和修复紫外线诱导的光损伤（例如，顺式-syn环丁烷嘧啶正餐，或CPDs）的主要候选参与者。事实上，HMGA1蛋白的过表达在体内和体外都抑制了CPDs的修复，并显著增加了细胞对紫外线诱导的杀伤的敏感性，这是切除修复缺陷细胞的特征。我们假设HMGA1蛋白至少通过两种方式抑制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）：(a)通过物理影响DNA和染色质底物中病变形成和去除的过程；(b)特异性NER和BER修复基因的转录抑制。本研究的具体目的是：(1)确定HMGA1蛋白在细胞内浓度可以通过实验调节的细胞以及HMGA1水平自然存在差异的细胞中，全基因组和基因特异性DNA损伤（如CPDs和烷基化碱基）的NER和BER的效率；(2)利用遗传互补等体内方法，区分HMGA1过表达对切除修复基因转录的抑制作用以及对其他NER和BER过程的抑制作用；(3)测定体外核小体组装前后合成DNA底物特定位点的DNA损伤（如CPDs或尿嘧啶）对HMGA1与其同源DNA序列结合的影响；(4)使用爪蟾卵母细胞核修复提取物（NER）或纯化的人蛋白（BER），测量结合的HMGA1蛋白对核小体组装前后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基因是唯一已知的致癌基因，编码真正的染色体结构蛋白，其过度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损伤修复的机制，将为了解基因突变积累和基因组不稳定发生的潜在原因提供重要的新见解，这些都是癌细胞的标志。这些研究还可能导致鉴定新的分子靶点，用于治疗许多人类恶性肿瘤。

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.