Role of HMGA1 proteins in DNA damage and excision repair

描述（由申请人提供）：哺乳动物HMGA 1染色质蛋白是结构转录因子，其特异性结合富含AT的DNA区域的小沟，从而使其成为参与识别和修复UV诱导的光损伤（例如，顺式-顺式环丁烷嘧啶二聚体或CPD）。 事实上，HMGA 1蛋白的过表达抑制CPD在体内和体外的修复，并且还显著增加细胞对UV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HMGA 1蛋白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）至少以两种方式：（a）通过物理影响DNA和染色质底物中损伤形成和去除的过程;和（B）通过特异性NER和BER修复基因的转录抑制。 本研究的具体目的是：（1）在细胞内HMGA 1蛋白浓度可以通过实验调节的细胞中以及在HMGA 1水平天然存在差异的细胞中，确定DNA损伤（如CPD和烷基化碱基）的全局基因组和基因特异性NER和BER的效率;（2）采用遗传互补和其他体内方法来区分HMGA 1过表达对切除修复基因转录的抑制和对其他NER和BER过程的抑制的影响;（3）在体外核小体组装之前和之后，测量合成DNA底物中特定位点处的DNA损伤（例如CPD或尿嘧啶）对HMGA 1与其同源DNA序列结合的影响;并且，在本发明中，（4）测量结合的HMGA 1蛋白对其含有位点-使用爪蟾卵母细胞核修复提取物（NER）或纯化的人蛋白（BER）在核小体组装前后的特异性损伤。 HMGA基因是唯一已知的编码真正的染色体结构蛋白的癌基因，其过度表达被认为是许多人类癌症的诊断特征。 因此，HMGA蛋白抑制DNA损伤修复的机制的阐明将提供重要的新的见解的潜在原因的积累的基因突变，基因组不稳定性的发生，这是癌细胞的标志。 这些研究还可能导致鉴定用于治疗许多人类恶性肿瘤的新分子靶点。

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.