Characterizing an Endonuclease Ensemble in Meiotic Crossing Over

减数分裂交叉中核酸内切酶整体的表征

• 批准号: 9119039
• 负责人: Carol M Manhart
• 金额: $ 5.8万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-01 至 2017-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9119039
• 关键词: Address; Binding; Biochemical; Bloom Syndrome; C-terminal; Chemistry; Chromosome Segregation; Chromosomes; Colorectal Cancer; Complex; Cruciform DNA; DNA; DNA biosynthesis; DNA-Directed DNA Polymerase; DNA-Protein Interaction; Data; Defect; Development; Diagnostic; Disease; Disease model; Early Diagnosis; Electrophoretic Mobility Shift Assay; Exonuclease; Fellowship; Frameshift Mutation; Genes; Genetic; Genetic Crossing Over; Genetic Recombination; Health; Hereditary Nonpolyposis Colorectal Neoplasms; Homologous Gene; Human; In Vitro; Individual National Research Service Award; Infertility; Inherited; Maps; Meiosis; Meiotic Recombination; Metals; Minor; Mismatch Repair; Modeling; Mus; Mutation; Nature; Oligonucleotides; Organism; Pathway interactions; Plasmids; Play; Polymerase; Positioning Attribute; Process; Protein-Protein Interaction Map; Proteins; Recruitment Activity; Research; Resolution; Role; Saccharomyces cerevisiae; Series; Signal Transduction; Site; Specificity; Structure; Substrate Specificity; Superhelical DNA; System; Testing; Time; Work; Yeasts; base; crosslink; drug development; drug discovery; endonuclease; genome integrity; helicase; improved; in vitro Assay; in vitro activity; in vivo; insertion/deletion mutation; mutant; novel; nuclease; plasmid DNA; prevent; protein function; repaired; research study; tool

DESCRIPTION (provided by applicant): DNA mismatch repair (MMR) proteins recognize and excise polymerase errors such as single base misincorporation and insertion/deletion loops during DNA replication. MMR factors also play an important role in meiotic recombination. Mutations in genes encoding MMR and meiotic recombination machinery, have been implicated in a variety of diseases. Understanding the pathways in which these proteins function biochemically will provide mechanistic models that can be used for drug discovery and the development of diagnostic tools. Mlh1-Mlh3 complex is a vital component in a pathway where crossovers form between homologous chromosomes during meiosis. This complex also interacts with Msh2-Msh3 to repair frameshift mutations misincorporated by DNA polymerase. The biochemical steps carried out by Mlh1-Mlh3 in either process are unknown and will be studied here in Saccharomyces cerevisiae, baker's yeast. The first step of meiotic recombination is the formation of double strand breaks, a portion of which are converted into double Holliday junctions (dHJs), which are resolved into crossovers (COs). In S. cerevisiae, genetic evidence suggests dHJ resolution is carried out by Msh4-Msh5, Sgs1 helicase, Exo1 XPG nuclease, and putative Mlh1-Mlh3 endonuclease activitiy. How Mlh1-Mlh3 functions as an endonuclease and how this activity can be used to resolve dHJs is unknown and is the major focus of this project. Our lab's initial characterization of purified yeast Mlh1-Mlh3 complex shows that it is a metal-dependent endonuclease and can nick supercoiled and open circle plasmid DNA. I observed stimulation by Msh2-Msh3, but not ATP or RFC/PCNA. In Aim 1, I will expand on experiments I performed since arriving in the Alani lab to identify the mechanism by which Msh2-Msh3 is stimulating Mlh1-Mlh3. I will also determine if Mlh1-Mlh3 can be strand-directed by the same mechanism as Mlh1-Pms1, the major endonuclease in MMR. Such an activity is a requirement for resolving dHJs into COs. I will also further characterize Mlh1-Mlh3's in vitro substrate requirements. In Aim 2, I will test a proposed dHJ resolution complex involving purified Msh4-Msh5, Sgs1, Exo1, and Mlh1-Mlh3 on model substrates based on the specifications determined in Aim 1. I will also use photo-crosslinking to map protein-protein and protein- DNA contacts as a dHJ resolution complex is assembled. Together these data will allow me to construct a detailed model of how dHJs are resolved into COs in vivo.

描述（由申请人提供）：DNA不匹配修复（MMR）蛋白在DNA复制过程中识别和消除聚合酶误差，例如单个碱基掺杂和插入/缺失环。 MMR因素在减数分裂重组中也起着重要作用。编码MMR和减数分裂重组机制的基因突变与多种疾病有关。了解这些蛋白质在生化上功能的途径将提供可用于药物发现和开发诊断工具的机械模型。 MLH1-MLH3复合物是在减数分裂过程中同源染色体之间跨界形成的途径中的重要组成部分。该复合物还与MSH2-MSH3相互作用，以修复由DNA聚合酶掺入的移码突变。 MLH1-MLH3在这两个过程中进行的生化步骤尚不清楚，并将在贝克的酵母菌酿酒酵母中进行研究。减数分裂重组的第一步是形成了双链断裂，其中一部分被转换为双霍利迪连接（DHJ），这些结合被分解为跨界车（COS）。在酿酒酵母中，遗传证据表明，MSH4-MSH5，SGS1解旋酶，EXO1 XPG核酸酶和推定的MLH1-MLH3内核酸酶激活进行了DHJ分辨率。 MLH1-MLH3如何用作核酸内切酶，以及该活动如何用于解决DHJ是未知的，并且是该项目的主要重点。我们实验室对纯化的酵母MLH1-MLH3复合物的初始表征显示 它是一种依赖金属的核酸内切酶，可以划痕超螺旋并打开圆形质粒DNA。我观察到MSH2-MSH3刺激，但未观察到ATP或RFC/PCNA。在AIM 1中，我将扩展自到达Alani实验室以来进行的实验，以识别MSH2-MSH3刺激MLH1-MLH3的机制。我还将确定MLH1-MLH3是否可以通过MLH1-PMS1（MMR中的主要核酸酶）相同的机制将其链定向。这样的活动是将DHJ解析为COS的要求。我还将进一步表征MLH1-MLH3的体外底物要求。在AIM 2中，我将根据AIM 1中确定的规格来测试涉及纯化的MSH4-MSH5，SGS1，EXO1和MLH1-MLH3的DHJ分辨率复合物。我还将使用Photo-Crosslinking来映射蛋白质 - 蛋白质 - 蛋白质蛋白质和蛋白质 - 蛋白质 - DNA接触dhj backents a dhj complects complectss组成。这些数据共同使我能够构建一个详细的模型，即如何将DHJ解析到体内COS中。