Characterizing an Endonuclease Ensemble in Meiotic Crossing Over

描述（由申请人提供）：DNA错配修复（MMR）蛋白识别并切除聚合酶错误，如DNA复制过程中的单碱基错误掺入和插入/缺失环。MMR因子在减数分裂重组中也起重要作用。编码MMR和减数分裂重组机制的基因突变与多种疾病有关。了解这些蛋白质的生物化学功能的途径将提供可用于药物发现和诊断工具开发的机制模型。Mlh 1-Mlh 3复合体是减数分裂过程中同源染色体之间形成交叉的途径中的重要组分。该复合物还与Msh 2-Msh 3相互作用以修复被DNA聚合酶错误掺入的移码突变。Mlh 1-Mlh 3在任一过程中进行的生化步骤是未知的，将在这里在酿酒酵母（Saccharomyces cerevisiae）中进行研究。减数分裂重组的第一步是形成双链断裂，其中一部分被转化为双霍利迪连接（dHJ），其被解析为交换（CO）。In S.在酿酒酵母中，遗传证据表明dHJ的拆分是通过Msh 4-Msh 5、Sgs 1解旋酶、Exo 1 XPG核酸酶和推定的Mlh 1-Mlh 3内切核酸酶活性进行的。Mlh 1-Mlh 3如何作为核酸内切酶发挥作用以及这种活性如何用于解析dHJ尚不清楚，这是该项目的主要焦点。我们实验室对纯化的酵母Mlh 1-Mlh 3复合物的初步表征表明， 它是一种金属依赖性核酸内切酶，可以切割超螺旋和开环质粒DNA。我观察到Msh 2-Msh 3的刺激，但没有ATP或RFC/PCNA。在目标1中，我将扩展我在Alani实验室进行的实验，以确定Msh 2-Msh 3刺激Mlh 1-Mlh 3的机制。我还将确定Mlh 1-Mlh 3是否可以通过与Mlh 1-Pms 1（MMR中的主要内切核酸酶）相同的机制进行链定向。这种活动是将dHJ解析为CO的要求。我还将进一步表征Mlh 1-Mlh 3的体外底物要求。在目标2中，本人将根据目标1中确定的质量标准，在模型底物上检测涉及纯化Msh 4-Msh 5、Sgs 1、Exo 1和Mlh 1-Mlh 3的拟定dHJ分离度复合物。我还将使用光交联来映射蛋白质-蛋白质和蛋白质- DNA接触，因为组装了dHJ解析复合物。这些数据将使我能够构建一个详细的模型，说明dHJ如何在体内分解为CO。

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.