Study of DNA repair mechanisms that affect chemosensitivity of breast cancer

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I have investigated the role of substrate ubiquitination by the breast and ovarian tumor suppressor BRCA1 on DNA damage response to elucidate the DNA damage repair mechanisms underlying chemosensitivity of breast cancer I previously identified several substrates including nucleophosmin (NPM1/B23) and RPB8, a common subunit of RNA polymerases. I have mainly investigated RPB8 in 2006 and found that 1) endogenous RPB8 interacts with BARD1, 2) RPB8 polyubiquitination by purified BRCAl-BARD1 in vitro, 3) Lys6-linked ubiquitin polymer formation in the RPB8 ubiquitination, 4) inhibition of in vivo RPB8 ubiquitination after UV irradiation by siRNA knockdown of BRCA1, 5) destabilization of endogenous RPB8 protein in vivo by siRNA knockdown of BRCA1, 6) HeLa cell lines stably expressing a ubiquitin-resistant form of RPB8 exhibited UV hypersensitivity accompanied by up-regulated caspase activity and p53 phosphorylation at Ser15. In 2006 I have mainly investigated NPM1. I established a cell lines stably expressing NPM1 mutant that is incapable of being ubiquitinated by BRCA1 and investigated its biological significance. However I finally concluded that the cells did not express the phenotype caused by the deficiency of the ubiquitination. I then reconstituted an in vitro NPM1 ubiquitination system using only purified recombinant proteins to investigate the effect of NPM1 ubiquitination on histone chaperone activity of NPM1. I further created recombinant proteins including NPM1, BRCA1-BARD1 heterodimer, Ube2w, Ubc13, and Mms2 and established Lys63-linked NPM1 polyubiquitination system to study DNA damage response.