| Summary: | 碩士 === 國立成功大學 === 生物化學研究所 === 88 === RPA ( replication protein A ) is a ubiquitous eukaryotic single-stranded DNA binding protein complex, that is composed of RPA1, RPA2 and RPA3 subunits. The heteraotrimeric complex has high affinity for single stranded and damaged DNA, and plays key roles in DNA replication, repair and recombination. Besides, RPA has been found to interact with several proteins. RAP1 can interact with SV40 large T antigen, DNA polymerase α, VP16 and p53, and RPA2 can interact with XPA, RAD52 and Uracil-DNA glycosylase which are involved in DNA repair and recombination. RPA2 is found to be phosphorylated in a cell cycle dependent manner, with phosphorylation of RPA2 seen at the G1/S transition and continuing through late M phase. In addition, RPA2 become hyperphosphorylated in response to DNA -damaging agents, such as UV or ionizing irradiation, and treatment with replication inhibitor. And the phosphorylation is carried out by DNA-dependent protein kinase ( DNA-PK ) , which is important for the processes of DNA repair and recombination. Recent studies indicate that DNA-PK phosphorylates RPA2 in the response of replication-mediated DNA damage, which may signal the presence of DNA damage to an S-phase checkpoint mechanism. The phosphorylation of RPA2 also prevents the association of RPA with p53. Therefore, RPA phosphorylation appears to act in cellular signaling pathway and may play an important role in DNA metabolism and the regulation of cell cycle. But the physiological role of phosphorylation of RPA is not clear.
To identify other cellular factors that can interact with RPA2, we have used yeast-two-hybrid system. About 30 positive clones were sequenced and alignment to NCBI database. Two clones that contained sequences identical to the almost full length of RPA3 and ribosomal protein L5 were identified respectively. RPA3 has been known to associate with RPA2 strongly whereas L5 is an 894bp, 34KDa ribosomal protein that is a component of 60S ribosome. L5 is responsible for 5SrRNA targeting to nucleolus. L5 can also be associated with type I phosphatase, CKII ( casein kinase II ) and oncoprotein mdm2. The results of cotransformation experiment and quantitativeβ- galactosidase assays using yeast-two-hybrid systems suggested that L5 is associated with RPA2. Radiolabled proteins were prepared by coupled in vitro transcription / translation and were subjected to immunoprecipitation analysis with anti-RPA2 antibody. This result revealed that L5 protein is associated with RPA2.A segment of L5 protein ( L5d 71-152 ) whose codon can be used easily in prokaryotic system was constructed. The L5d 71-152 gene product was overexpressed in E. coli, purified, and the anti-L5d 71-152 antibody was obtained. The interaction of L5 with RPA2, especially with which form ( hypo-, hyper-, or dephosphorylated ) of RPA2, in the cell will be further investigate.
|
|---|
