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. ? ?
