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，它对所有生物体的生存至关重要。负责启动错配修复的蛋白质MutS和MutL同源物在整个真核生物中高度保守;而下游修复事件的保守性较低。Drokaryot.es MutS和MutL同源物是二聚体蛋白，其含有DNA结合和ATP酶活性，这对于体内MMR是必需的。MMR是由MutS同源物与错配结合启动的。随后，MutL同源物以ATP依赖性方式与MutS同源物相互作用，并协调蛋白质-蛋白质相互作用，其信号切除和重新合成含有不正确核苷酸的新合成的DNA链。最近，真核生物的双向错配修复已经在体外使用含有切口的错配DNA重建，并且除了MutS和MutL蛋白之外，它还需要核酸外切酶Exo 1（尽管可能涉及其他）、钳装载蛋白RFC和钳蛋白PCNA、DNA聚合酶6，并且在单链结合蛋白RFA和HMGB 1存在下修复增强。在人类中，MutS和MutL同源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。为了了解这些突变如何导致错配修复缺陷，有必要阐明MMR的分子机制，并确定突变如何改变该机制。生物化学研究表明，蛋白质和蛋白质-DNA复合物的不同构象状态对MMR的调节至关重要。为了表征这些复合物，我们将使用原子力显微镜（AFM），它提供了一个很好的方法，我们可以直接观察到这种复合物的构象性质的变化。从一组AFM实验，我们可以确定的结合亲和力，特异性和化学计量，以及蛋白质-DNA复合物的构象特性。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和缔合常数。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。作为原子力显微镜研究的补充，我们将使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的结合和蛋白质诱导的DNA在溶液中的弯曲。我们的长期目标是组装具有完全DNA修复功能的复合物;然而，在这项研究中，我们专注于参与真核MMR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.