Role of HMGA1 proteins in DNA damage and excision repair

描述(申请人提供)：哺乳动物的HMGA1染色质蛋白是一种结构性转录因子，能与富含AT的DNA区域的细小凹槽特异结合，从而使它们成为识别和修复紫外线诱导的光损伤(例如顺式环丁烷嘧啶晚餐，或CPD)的主要候选者。事实上，HMGA1蛋白的过表达在体内和体外都抑制了CPD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局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的形成和去除过程；以及(B)通过转录抑制特定的NER和BER修复基因。这项研究的具体目的是：(1)确定DNA损伤(如CPDS和烷化碱基)的全局基因组和基因特异性NER和BER的效率，在细胞内HMGA1蛋白浓度可以实验调节的细胞中，以及在HMGA1水平自然存在差异的细胞中；(2)利用遗传互补和其他体内方法区分HMGA1过表达对抑制切除修复基因转录和抑制其他NER和BER过程的影响；(3)测量合成DNA底物中特定位置的DNA损伤，如CPDS或尿嘧啶，在体外核小体组装前后对HMGA1与其同源DNA序列结合的影响；以及(4)使用非洲爪哇卵母细胞核修复提取物(NER)或纯化的人类蛋白(BER)，测量结合的HMGA1蛋白在核小体组装前后对其包含位置特异性损伤的同源DNA结合序列的NER或BER的影响。HMGA基因是已知的唯一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，阐明HMGA蛋白抑制DNA损伤修复的机制将为深入了解基因突变积累和基因组不稳定性的潜在原因提供重要的新见解，这些都是癌细胞的特征。这些研究还可能导致识别新的分子靶点，用于治疗一些人类恶性肿瘤。

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.