Structure Function Studies of DNA Mismatch Repair

DNA错配修复的结构功能研究

• 批准号: 7898837
• 负责人: DOROTHY A ERIE
• 金额: $ 29.61万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-01 至 2013-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7898837
• 关键词: ATP phosphohydrolase; Affinity; Atomic Force Microscopy; Binding; Binding Proteins; Biochemical; Complement; Complex; DNA; DNA Binding; DNA Repair; DNA biosynthesis; DNA-Directed DNA Polymerase; Defect; Discrimination; Escherichia coli; Escherichia coli Proteins; Eukaryota; Event; Excision; Exonuclease; Fluorescence Anisotropy; Fluorescence Resonance Energy Transfer; Goals; Grant; HMGB1 gene; Hereditary Nonpolyposis Colorectal Neoplasms; Homologous Gene; Human; Hydrolysis; Image; In Vitro; Link; Malignant Neoplasms; Mediating; Methods; Mismatch Repair; Molecular; Molecular Conformation; Mutation; Nucleotides; Organism; Polymerase; Process; Prokaryotic Cells; Property; Protein Binding; Protein Dynamics; Proteins; Regulation; Research Personnel; Role; Signal Transduction; Site; Solutions; Specificity; Structure; Structure-Activity Relationship; System; cofactor; dimer; endonuclease; exonuclease II; in vivo; mutant; programs; protein complex; protein protein interaction; reconstitution; repaired; research study; stoichiometry

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.

描述(由申请人提供)：本提案的主要目标是阐明支配真核DNA错配修复的结构-功能关系。DNA错配修复(MMR)是DNA合成错误被复制后纠正的机制，它对所有生物的生存都是至关重要的。负责启动错配修复的蛋白质MutS和MutL在真核生物和真核生物中高度保守，而下游的修复事件则不太保守。MutS和MutL lomlos是二聚体蛋白，既含有DNA结合活性，又含有体内MMR所必需的ATPase活性。MMR是由MutS同源基因与错配结合而启动的。随后，MutL同源物以依赖于ATP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，从而发出信号，切除和重新合成含有错误核苷酸的新合成的DNA链。最近，利用含有缺口的错配DNA在体外重组了真核细胞的双向错配修复，除了MutS和MutL蛋白外，它还需要外切酶Exo1(尽管可能涉及其他蛋白质)、钳制加载蛋白RFC和钳制蛋白PCNA、DNA聚合酶6，并且修复在单链结合蛋白RFA和HMGB1的存在下得到加强。在人类中，MutS和MutL同源基因的突变与遗传性非息肉病性结直肠癌(HNPCC)直接相关，并与散发性癌症有关。为了了解这些突变是如何导致错配修复中的缺陷的，有必要阐明MMR的分子机制并确定突变是如何改变该机制的。生化研究表明，蛋白质和蛋白质-DNA复合体的不同构象状态是MMR调控的中心。为了表征这些络合物，我们将使用原子力显微镜(AFM)，它提供了一种很好的方法，通过它我们可以直接观察这些络合物的构象性质的变化。通过一组AFM实验，我们可以确定蛋白质-DNA复合体的结合亲和力、特异性和化学计量以及构象性质。此外，我们还可以表征单个蛋白质的构象变化，并确定蛋白质-蛋白质复合体的化学计量和结合常数。最后，我们可以使用溶液成像来跟踪蛋白质-DNA复合体的动力学。作为对AFM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DNA修复功能的复合体；然而，在本研究中，我们重点研究了几个参与真核MMR的蛋白质-蛋白质和蛋白质-DNA复合体的结构和功能。