Structural Studies Of DNA Recombination, Repair, and Rep

DNA易受各种突变和化学修饰的影响。DNA复制过程中的错误，无论是错配对还是滑移，都会导致碱基对不匹配，其发生频率为10-8至10-6。暴露于紫外线照射或化学试剂可导致共价修饰的DNA碱基，程序性减数分裂和有丝分裂DNA重排，电离辐射和氧化剂可导致双链DNA断裂。为了保持基因组的完整性和维持生命，细菌、古细菌和真核生物使用保守的机制来修复或容忍每种类型的损伤。我的研究小组继续进行大肠杆菌和人类错配修复过程以及病变旁路DNA合成的结构和功能研究。

DNA is susceptible to a variety of mutations and chemical modifications. Errors during DNA replication, either mispairing or slippage, result in mismatched base pairs, which occur at a frequency of 10-8 to 10-6. Exposure to UV irradiation or chemical agents may lead to covalently modified DNA bases, and programmed meiotic and mitotic DNA rearrangement, ionizing radiation and oxidative agents can result in double-strand DNA breaks. To maintain genomic integrity and to sustain life, bacteria, archaea and eukarya use conserved mechanisms to repair or to tolerate each type of damage. My research group has continued to carry on structural and functional studies of E. coli and human mismatch repair processes and lesion-bypass DNA synthesis. Mismatch repair in E. coli is initiated by three proteins, MutS, MutL and MutH, to specifically target the newly synthesized daughter strand. MutS is an ATPase and recognizes a mismatched base-pair as well as an insertion or deletion of 1-4 nucleotides in one strand. MutH is a latent endonuclease that is both sequence- and methylation-specific; when activated by MutS upon detection of a mismatch, it cleaves 5? to the unmethylated d(GATC) sequence in a hemimethylated duplex, thus targeting mismatch repair to newly synthesized and unmethylated daughter strand. MutL mediates the communication between MutS and MutH, which do not directly interact. Once a nick is introduced in the daughter strand by MutH, DNA exonuclease, UvrD helicase, single-strand binding protein and DNA polymerase III are recruited to remove nucleotides from the nick to beyond the mismatch and to fill in the resulting gap. Homologues of MutS and MutL are found in all eukaryotes, and malfunction of either human MutS or MutL homolog is directly implicated in the susceptibility to hereditary non-polyposis colorectal cancer (HNPCC) and other sporadic cancers. After determining the crystal structures of MutS, MutL and MutH between 1998 and 2001, we have constructed 47 structure-based mutations of MutS, MutL and MutH and analyzed the function of each protein in a series of in vitro assays including DNA binding, ATPase activity, DNA cleavage and mismatch repair initiation. In collaboration with Dr. Jeffrey Miller at UCLA, we have also characterized mutation and recombination rates of E. coli cells expressing the mutant proteins in vivo. Results from the mutant studies not only support the mismatch repair model we proposed that MutS stays bound to the mismatch site while activating the repair process but also illuminate the coordination between mismatch repair and prevention of homologous recombination between similar but not identical DNA sequences. Lesion-bypass DNA synthesis is carried out by the recently discovered Y-family DNA polymerases, which perform low-fidelity synthesis on undamaged DNA templates and are able to traverse normally replication-blocking lesions, including abasic sites, 8-oxo-G, benzopyrene adducts, and cyclobutane pyrimidine dimmers. Y-family polymerases are widespread and enable species from E. coli to human to tolerate UV irradiation and various forms of base modification. Each individual Y-family polymerase exhibits a distinct substrate preference. For example, Pol h is particularly efficient to bypass the UV crosslinking product, cyclobutane pyrimidine dimers. Mutations in XPV, which encodes human Pol h, are correlated to 20% of xeroderma pigmentosum. After publishing the first Y-family polymerase and DNA complex structure in 2001, we have recently determined the crystal structures of an archaeal Y ?family polymerase, Dpo4, complexed with a cyclobutane pyrimidine dimers. Our structures suggest a mechanism by which specific Y-family polymerases are able to bypass a thymine dimer while replicative DNA polymerases cannot. We are continuing to study structure of multiprotein and nucleic acid complexes involving in DNA repair and replication to elucidating molecular mechanism that underlies in human diseases.