Study of DNA repair mechanisms that affect chemosensitivity of breast cancer

为了阐明乳腺癌化疗敏感性的DNA损伤修复机制，我研究了乳腺和卵巢肿瘤抑制因子BRCA 1对DNA损伤反应的底物泛素化作用。我以前鉴定了几种底物，包括核磷蛋白（NPM 1/B23）和RNA聚合酶的常见亚基RPB 8。本人在2006年主要研究了RPB 8，发现1）内源性RPB 8与BARD 1相互作用，2）RPB 8在体外被纯化的BRCA 1-BARD 1多聚泛素化，3）在RPB 8泛素化过程中形成Lys 6连接的泛素聚合物，4）通过siRNA敲低BRCA 1抑制UV照射后的体内RPB 8泛素化，5）通过BRCA 1的siRNA敲低使内源性RPB 8蛋白在体内不稳定。6）稳定表达RPB 8的泛素抗性形式的HeLa细胞系表现出UV超敏性，伴随着上调的半胱天冬酶活性和p53在Ser 15处的磷酸化。2006年，我主要研究了NPM 1。建立了稳定表达不能被BRCA 1泛素化的NPM 1突变体的细胞系，并研究了其生物学意义。然而，我最终得出结论，细胞不表达由泛素化缺陷引起的表型。然后，仅使用纯化的重组蛋白重建体外NPM 1泛素化系统，以研究NPM 1泛素化对NPM 1的组蛋白伴侣活性的影响。进一步构建了NPM 1、BRCA 1-BARD 1异源二聚体、Ube 2 w、Ubc 13和Mms 2等重组蛋白，并建立了Lys 63连接的NPM 1多聚泛素化系统来研究DNA损伤反应。

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.