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Molecular Mechanisms of Human Homologous Recombination

人类同源重组的分子机制

基本信息

批准号：
10641844
负责人：
John Brooks Crickard
金额：
$ 37.52万
依托单位：
CORNELL UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-08-12 至 2026-06-30
项目状态：
未结题

项目摘要

SUMMARY/ABSTRACT Organisms are constantly exposed to environmental conditions that challenge the integrity of the genome. Loss of genomic integrity contributes to the development of most cancers. DNA double strand breaks (DSBs) are a dangerous type of DNA damage that can lead to rapid loss of sequence information stored within the genome. Homologous recombination (HR) is one of the primary DSB repair pathways and is predicated on locating an undamaged DNA sequence that matches the damaged DNA sequence elsewhere in the genome. The homologous sequence can then be used to restore the lost DNA sequence information. During normal mitotic growth, HR preferentially repairs DSBs using sequence information stored in the sister chromatid. Aiding in maintenance of allelic variation between genes and preventing unbalanced exchange of genetic information between chromosomes. In contrast during meiosis the homologous chromosome becomes the preferred DNA repair substrate. There is a large amount information on existing pathways that have evolved in S. cerevisiae to promote DNA repair from the homologous chromosome during meiosis. However, little is known about how homologous chromosomes are used for repair in humans. One of the key determinants in chromosome choice during HR, is the organization of the presynaptic complex (PSC). The regulation, formation, and activity of the human PSC is controlled by >45 proteins. However, a basic functional unit of the PSC consists of RAD51 and associated factors (RAD54L) during mitosis, and RAD51, DMC1 and their associated factors (RAD54L, RAD54B, HOP2-MND1) during meiosis. Understanding how these proteins organize into active complexes during HR is a critical step in understanding how human homologous chromosomes are used for HR. Over the course of our studies we will use biochemical and single molecule approaches to understand the mechanism behind RAD51 and DMC1 self-segregation during meiotic PSC formation. We will understand how DMC1 forms a meiotic homology search complex, and with cooperation of accessory proteins, aligns DNA sequences. We will identify how meiotic homology search complexes overcome chromatin. Finally, we will work to understand how conflicts between the two highly related motor protein RAD54L and RAD54B may promote homologous chromosome use during human mitotic HR. In summary, the primary goal of this research proposal will be to use molecular biology, biochemistry, and single molecule approaches to understand how human mitotic and meiotic PSCs organize, and promote DNA sequence alignment during HR. The data we collect from these experiments will be used to build a model for how human homologous chromosome selection may occur during both mitotic and meiotic HR.

摘要/摘要生物体不断暴露在挑战基因组完整性的环境条件下。基因组完整性的丧失导致大多数癌症的发生。 DNA 双链断裂 (DSB) 是一种危险的 DNA 损伤，可能导致存储在 DNA 中的序列信息快速丢失。基因组。同源重组 (HR) 是主要的 DSB 修复途径之一，其基础是定位与基因组中其他地方受损的 DNA 序列相匹配的未受损的 DNA 序列。然后可以使用同源序列来恢复丢失的DNA序列信息。正常期间有丝分裂生长时，HR 优先使用姐妹染色单体中存储的序列信息修复 DSB。帮助维持基因之间的等位基因变异并防止遗传的不平衡交换染色体之间的信息。相反，在减数分裂期间，同源染色体变成优选的DNA修复底物。有大量关于现有途径的信息，这些信息已在酿酒酵母在减数分裂过程中促进同源染色体的 DNA 修复。然而，很少的是了解同源染色体如何用于人类修复。的关键决定因素之一 HR 期间的染色体选择是突触前复合体 (PSC) 的组织。该规定，人类 PSC 的形成和活性由超过 45 种蛋白质控制。然而，一个基本的功能单元 PSC 由 RAD51 和有丝分裂期间的相关因子 (RAD54L) 组成，RAD51、DMC1 及其减数分裂期间的相关因素（RAD54L、RAD54B、HOP2-MND1）。了解这些蛋白质如何在 HR 过程中组织成活性复合物是理解人类同源性如何形成的关键一步染色体用于 HR。在我们的研究过程中，我们将使用生化和单分子了解减数分裂 PSC 期间 RAD51 和 DMC1 自分离机制的方法形成。我们将了解 DMC1 如何形成减数分裂同源搜索复合体，并与辅助蛋白，对齐 DNA 序列。我们将确定减数分裂同源性搜索复合体如何克服染色质。最后，我们将努力了解这两个高度相关的运动之间的冲突是如何发生的蛋白质 RAD54L 和 RAD54B 可能促进人类有丝分裂 HR 期间同源染色体的使用。在总之，本研究计划的主要目标是利用分子生物学、生物化学和单一方法分子方法来了解人类有丝分裂和减数分裂 PSC 如何组织和促进 DNA HR 期间的序列比对。我们从这些实验中收集的数据将用于构建模型人类同源染色体选择如何在有丝分裂和减数分裂 HR 期间发生。