Functional studies of the human hHR23A and hHR23B proteins in DNA repair and proteolysis

• Main Authors: Yu-Hsin Huang, 黃聿忻
• Other Authors: Wenya Huang
• Format: Others
• Language: zh-TW
• Published: 2003
• Online Access: http://ndltd.ncl.edu.tw/handle/qfcf9g

碩士 === 國立成功大學 === 分子醫學研究所 === 91 === Human hHR23A and hHR23B genes are the homologues of Saccharomyces cerevisiae RAD23 gene and these two factors both contain functional domains including an ubiquitin-like (UBL) domain at the N-termini, two ubiquitin-associated (UBA) domains and an XPC-binding domain. Previous studies showed that the hHR23B protein is complexed with the XP group C (XPC) protein, defective in the xeroderma pigmentosum C syndrome, and is involved in the global genome nucleotide excision repair (NER) pathway, but the functions of hHR23A remain to be elucidated. We found that the XPC-binding domain of hHR23B contributes to a dominant negative phenotype for the wild-type hHR23B protein and partially inhibits the function of repair system by the host cell reactivation (HCR) assay. On the contrary, the XPC-binding domain of hHR23A didn’t exhibit such an effect. It indicated that only hHR23B participates in global genome repair (GGR) pathway, but hHR23A does not. These data have been confirmed in NIH/3T3 cells stably transfected with the XPC-binding domain of the hHR23B. In past years, RNA interference (RNAi) system has been a powerful tool to establish knock-down models in vitro and in vivo. We constructed three RNAi constructs of each of the hHR23A or hHR23B genes, attempting to clarify the functions of hHR23A/hHR23B. In our data, two of the hHR23A and one hHR23B constructs inhibited endogenous levels of the hHR23A or hHR23B in HtTA1 cells, assayed by Western blot. These data have been confirmed by RT-PCR. In addition, the UBA domains of hHR23A/hHR23B are presumably ubiquitinated. The mutants in potential ubiquitination sites on UBA domains of hHR23A will therefore be examined for their ubiquitination activities. Using these potential ubiquitination mutants, the hHR23A would be studied for their roles in proteolysis and DNA repair.