The protein interaction studies of the two humannucleotide excision repair factors hHR23A and hHR23Band their roles as protein degradation regulators

• Main Authors: Yeun-Tyng Lai, 賴允婷
• Other Authors: Wenya Huang
• Format: Others
• Language: zh-TW
• Online Access: http://ndltd.ncl.edu.tw/handle/29164176230006500969

碩士 === 國立成功大學 === 分子醫學研究所 === 90 === 　　DNA repair is the major cellular mechanism by which the DNA damages on the genome are removed. The nucleotide excision repair (NER) mainly operates on a large spectrum of base damages, particularly that produced by ultraviolet (UV) irradiation and carcinogenic chemicals which produce bulky, helix-distorting lesions in DNA structure. Xeroderma pigmentosum (XP) syndromes are the diseases that are caused by mutations in NER genes. Rad (radiation-sensitive) 23 is the NER factor in the yeast S. cerevisiae and may regulate NER pathway by associating with protein degradation. hHR23 (human homolog of Rad23) A and hHR23B are two sequence homologs of Rad23; however, their roles in NER in vivo remain to be elucidated. hHR23A and B are highly homologous with each other in that both of them contain the ubiquitin-like (UBL) domain at the N-termini and two ubiquitin-associated (UBA) domains at the C-termini. In the yeast two-hybrid screening assay using hHR23A/B as the bait, three proteins were identified: the 26S proteasome subunits (1) TRAP2 and (2) S5a, and (3) ubiquitin. 　　To study the intermolecular or intramolecular interactions through the UBL and UBA domains, we designed some hHR23A/B truncated forms and used yeast two-hybrid to assay. Our data have shown that hHR23A interacts with hHR23B even without the UBL domain of hHR23B. In addition, hHR23B and hHR23B may become homodimer. Because of the interactions of hHR23A/B with the 26S proteasome, we suggested hHR23A/B may be involved in protein degradation. By N-end rule, we designed a reporter gene which contains ubiquitin, different N-end DHFRts and beta-galactosidase to examine the protein degradation in vivo. The preliminary results of these reporters have shown the relationship with N-end rule, now we just test the influences of protein degradation inhibitor. Through experiments of the protein interaction and the protein degradation reporter system, we will understand more about the properties and functions of hHR23A and hHR23B.