Structural Studies Of DNA Recombination, Repair, and Rep

基因组DNA必须在每个细胞分裂周期之前进行复制。尽管复制DNA聚合酶具有内置的校对机制，以尽量减少复制过程中的错误，但偶尔会发生由于复制错误而导致的错配。错配修复系统，以防止突变的复制错误存在于大多数生物体。大肠杆菌具有甲基定向错配修复系统，包括MutS， MutL和MutH蛋白。在人类中也发现了MutS和MutL蛋白的同源物。在90%的遗传性非息肉病性结直肠癌中发现了MutS或MutL同系物的突变。到2001年10月，我们的团队确定了错配修复蛋白MutH的晶体结构，MutL的一个保守的40KD ATP酶片段及其核苷酸复合物，人类MutL同源物PMS2的ATP酶结构域，单独与ATP和不可水解的ATP类似物络合，以及190 Kd Taq MutS单独与DNA络合，并与DNA和ADP/Mg2+三元络合。去年（2001-2002），(1)基于晶体结构，我们构建了47个MutS， MutL和MutH大肠杆菌突变体，进行了体外和体内（与加州大学洛杉矶分校的J. Miller合作）的生化研究，发现每个不能修复错配的突变体都缺乏防止同源重组的能力。我们的研究还发现mut的非特异性DNA结合特性是mut切割DNA后错配修复所必需的，并且mut的结构域协同工作以实现异双工与同双工的最大差异结合，而不是最大的DNA结合。(2) MutL atp酶活性位点与DNA拓扑异构酶、Hsp90、细菌和线粒体激酶共享保守的序列基序。这些蛋白质中有许多是潜在的药物靶点。我们发现，尽管这些酶具有相似的ATP结合袋和对Mg离子的共同需求，但它们在ATP结合袋中的单价离子偏好不同。基于MutL与大鼠线粒体蛋白激酶的结构和序列比较，我们对MutL进行了点突变，将MutL从使用任何单态离子转化为特异性的Na+。我们还确定了野生型和突变型MutL蛋白的晶体结构，并观察到Na+对K+的交换。我们建议单价离子的特异性可以用于未来的药物设计。

Genomic DNA has to be replicated before every cycle of cell division. Although replicative DNA polymerases have a built-in proofreading mechanism to minimize errors during replication, occasionally mismatch due to replication-error occurs. Mismatch repair systems to prevent mutations from replicative errors exist in most organisms. E. coli has a methyl-directed mismatch repair system comprising MutS, MutL and MutH proteins. Homologues of MutS and MutL proteins are also found in humans. Mutations in MutS or MutL homologs have been identified in 90% of the hereditary nonpolyposis colorectal cancers. By Oct. 2001, our group determined the crystal structures of mismatch repair proteins MutH, a conserved 40KD ATPase fragment of MutL and its complexes with nucleotides, the ATPase domain of a human MutL homologue, PMS2, alone and complexed with ATP and nonhydrolyzeable ATP analog, and the 190 Kd Taq MutS alone, complexed with DNA, and as a ternary complex with DNA and ADP/Mg2+. Last year (2001-2002), (1) based on the crystal structures, we constructed 47 MutS, MutL and MutH E. coli mutants, carried out in vitro and in vivo (in collaboration with J. Miller at UCLA) biochemical studies, and found that every mutant that fails to repair mismatch is deficient in preventing homeologous recombination. Our studies also revealed that the non-specific DNA binding property of MutL is required for mismatch repair after the DNA incision by MutH and that the structural domains in MutS work cooperatively to achieve maximal differential binding of heteroduplex versus homoduplex rather than maximal DNA binding. (2) The MutL ATPase active site shares conserved sequence motifs with DNA topoisomerases, Hsp90 and bacterial and mitochondrial kinases. Many of these proteins are potential drug targets. We found that despite the similar ATP binding pocket among these enzymes and a shared requirment of Mg ion, each of them differs in monovalent ion preference in the ATP binding pocket. Based on structure and sequence comparison between MutL and a rat mitochondrial protein kinase, we made a point mutation in MutL and converted MutL from using any monomalent ion to Na+ specifically. We have also determined the crystal structures of the wildtype and mutant MutL proteins and observed a Na+ for K+ exchange. We propose that the monovalent ion specificity may be exploited for future drug design. We have also determined the crystal structure of a hemimethylated GATC binding proten, E. coli SeqA, bound to the DNA. This project was derived from our research on how MutH recognizes hemimethylated GATC sequence and targets mismatch repair to the daughter strand specifically. The structure of SeqA-DNA complex reveals that the recognition of the methylated adenine is achieved by protein mainchain atoms via close van der Waals contacts. Most interestingly, the structure suggests a mechanism for SeqA to sequestrate DNA replication origin, oriC, from being used prematurely, repeatedly or asynchronously. A new family of DNA polymerases, the Y-family, has recently been identified. They differ from the previously known DNA polymerases in the primary sequence and in the ability of lesion-bypass and error-prone DNA synthesis. In collaboration with Dr. Roger Woodgate of NICHD, we determined the crystal structure of a Y-family DNA polymerase, Dpo4 from S. Solfotaricus, in complex with undamaged DNA and an incoming nucleotide in 2001. These crystal structures provide the first atomic view of the Y-family polymerase in action and reveal a molecular mechanism for the low fidelity DNA synthesis and bypassing modified DNA bases. Recently, we have crystallized Dpo4 in complex with damaged or mispaired DNA. The new structures suggest that Dpo4 binds an incoming nucleotide independent of correct base pairing with the template strand and shuffles the template strand to find a partner for the incoming nucleotide before catalyzing the nucleotidyl-transfer reaction. We propose tht this "free-loading" of nucleotide serves Dpo4 to bypass DNA lesions as well as to make low fidelity DNA synthesis.