古菌RecJ-like核酸酶参与校正RNA引物3'末端错配核糖核苷酸的分子机制研究

古菌DNA聚合酶polB需要3'末端正确配对的RNA引物合成DNA，错配阻碍合成。我们发现古菌引物酶延伸RNA引物时，会在3'末端形成与DNA模板错配的核糖核苷酸。据此推测需修正RNA引物3'错配，以消除DNA延伸障碍。初步结果表明古菌P.furiosus编码的RecJ类似蛋白PfRecJ分别从3'和5'端水解RNA和DNA，并优先切除RNA/DNA杂合双链3'端错配的核糖核苷酸，可能负责校正RNA引物3'错配。为了进一步阐明古菌RNA引物3'错配校正机制及其生物学意义，本项目拟鉴定古菌PfRecJ酶学特性及其与细菌RecJ的异同，重点揭示PfRecJ对DNA和RNA水解极性差异、重构RNA引物3'错配校正反应；基因敲除手段鉴定嗜盐古菌H.volcanii recj-like基因的RNA引物错配校正功能；解析PfRecJ及其与核酸共结晶的三维结构。研究结果有助于加深认识古菌DNA复制机制。

Replicative DNA polymerases require a RNA primer for chromosomal DNA synthesis, wherein primase is responsible for the de novo synthesis of a complementary RNA primer. However, the archaeal primase from Pyrococcus furiosus often incorporates a mismatched nucleoside monophosphate, resulting in a 3'-mismatched RNA primer. Pfu DNA polymerase, an archaeal B-family DNA polymerase, cannot elongate the 3'-mismatched RNA primer because it cannot remove the 3'-mismatched ribonucleotide. Our primary biochemical results demonstrate the potential of a RecJ-like protein from P. furiosus (PfRecJ) in proofreading the 3'-mismatched ribonucleotides. Except for hydrolysis of single-stranded DNA in the 5'-3' direction, PfRecJ also hydrolyzes the single-stranded RNA and the RNA strand of RNA/DNA hybrids in the 3'-5' direction. Based on these results, we plan to further elucidate the possible mechanism and function of archaeal RecJ-like protein in proofreading 3'-mismatched RNA primer during chromosomal DNA replication. The research is focused on the following three aspects: 1) biochemical characterization of PfRecJ, including the mechanism in hydrolyzing RNA and DNA, biochemical comparison with bacterial RecJ, and in vitro reconsitituting the archaeal proofreading reaction of 3'-mismatched RNA primer; 2) identifying, in vivo, the function of recj-like gene in H. volcanii by deleting recj-like gene and assaying mutant growth phenotype; 3) the structural biology of P. furiosus RecJ-like protein, including crystal structure of PfRecJ and PfRecJ-nucleic acid complex. The results will give a full and detailed understanding on the funtion of RecJ-like protein in archaea.