Structure Function Studies of DNA Mismatch Repair

描述（由申请人提供）：本提案的主要目标是阐明控制真核DNA错配修复的结构-功能关系。DNA错配修复（MMR）是DNA合成错误在复制后得到纠正的机制，是所有生物生存的核心。负责启动错配修复的蛋白，包括MutS和MutL同源蛋白，在真核生物和原核生物中高度保守；而下游修复事件的保守性较差。MutS和MutL同源体是二聚体蛋白，包含DNA结合和atp酶活性，这是体内MMR所必需的。MMR是由MutS同源物结合到一个不匹配启动的。随后，MutL同源物以atp依赖的方式与MutS同源物相互作用，并协调蛋白质-蛋白质相互作用，信号切除和重新合成新合成的含有错误核苷酸的DNA链。最近，利用含有缺口的错配DNA在体外重建了真核生物的双向错配修复，它需要外切酶Exo1（尽管其他可能涉及）、夹紧装载蛋白RFC和夹紧蛋白PCNA、DNA聚合酶6以及MutS和MutL蛋白，并且在单链结合蛋白RFA和HMGB1的存在下，修复得到增强。在人类中，MutS和MutL同系物的突变与遗传性非息肉病性结直肠癌（HNPCC）直接相关，并与散发性癌症相关。为了理解这些突变是如何导致错配修复缺陷的，有必要阐明MMR的分子机制，并确定突变如何改变这一机制。生化研究表明，蛋白质和蛋白质- dna复合物的不同构象状态是MMR调控的核心。为了表征这些配合物，我们将使用原子力显微镜（AFM），它提供了一个很好的方法，我们可以直接观察这些配合物的构象性质的变化。从一组AFM实验中，我们可以确定蛋白质- dna复合物的结合亲和力、特异性和化学计量学，以及构象性质。此外，我们可以表征单个蛋白质的构象变化，并确定蛋白质-蛋白质复合物的化学计量学和结合常数。最后，我们可以使用溶液成像来跟踪蛋白质- dna复合物的动力学。作为AFM研究的补充，我们将利用荧光各向异性和荧光共振能量转移（FRET）来表征蛋白质与DNA的结合以及蛋白质在溶液中诱导的DNA弯曲。我们的长期目标是组装具有完整DNA修复功能的复合物；然而，在本研究中，我们重点研究了真核生物MMR中涉及的几种蛋白质-蛋白质和蛋白质- dna复合物的结构和功能。

DESCRIPTION (provided by applicant): The primary goal of this proposal is to elucidate the structure-function relationships that govern eukaryotic DNA mismatch repair. DNA mismatch repair (MMR) is the mechanism by which DNA synthesis errors are corrected post-replicatively, and it is central to the survival of all organisms. The proteins, MutS and MutL homologs, responsible for the initiation of mismatch repair are highly conserved throughout Drokaryot.es arid eukaryotes; while, the downstream repair events are less well conserved. MutS and MutL lomologs are dimeric proteins which contain both DNA binding and ATPase activities that are essential for MMR in vivo. MMR is initiated by MutS homologs binding to a mismatch. Subsequently, MutL homologs interact with the MutS homologs in an ATP-dependent manner and coordinate protein-protein interactions that signal excision and resynthesis of the newly synthesized DNA strand containing the incorrect nucleotide. Recently eukaryotic bidirectional mismatch repair has been reconstituted in vitro using mismatch DNA containing a nick, and it requires the exonuclease, Exo1 (although others may be involved), the clamp loader protein, RFC, and clamp protein, PCNA, DNA polymerase 6, in addition to MutS and MutL proteins, and repair is enhanced in the presence of the single-stranded binding protein, RFA, and HMGB1. In humans, mutations in the MutS and MutL homologs are directly linked to hereditary non-polyposis colorectal cancer (HNPCC) and are associated with sporadic cancers. To understand how such mutations cause defects in mismatch repair, it is necessary to elucidate the molecular mechanisms of MMR and determine how mutations alter the mechanism. Biochemical studies indicate that different conformational states of the proteins and protein-DNA complexes are central to the regulation of MMR. To characterize these complexes, we will use atomic force microscopy (AFM) which provides an excellent method by which we can directly observe changes in conformational properties of such complexes. From a single set of AFM experiments, we can determine the binding affinity, specificity, and stoichiometry, as well as the conformational properties of the protein-DNA complexes. In addition, we can characterize conformational changes in single proteins and determine stoichiometries and association constants of protein-protein complexes. Finally, we can follow the dynamics of the protein-DNA complexes using solution imaging. As a complement to the AFM studies, we will use fluorescence anisotropy arid fluorescence resonance energy transfer (FRET) to characterize protein binding to DNA and protein-induced DNA bending in solution. Our long-term goal is to assemble complexes that are fully functional for DNA repair; however, in this study, we focus on the structure and function of several of the protein-protein and protein-DNA complexes that are involved in eukaryotic MMR.