Molecular Mechanisms of Human Homologous Recombination

摘要/摘要 生物体不断地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了大多数癌症的发生。DNA双链断裂(DSB) 是一种危险的DNA损伤类型，可能导致存储在 基因组。同源重组(HR)是DSB修复的主要途径之一 定位未受损的DNA序列，该序列与基因组中其他位置的受损DNA序列匹配。 然后可以使用同源序列来恢复丢失的DNA序列信息。在正常期间 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。 有助于保持基因间的等位变异，防止基因的不平衡交换 染色体之间的信息。相反，在减数分裂过程中，同源染色体变成 首选DNA修复底物。关于进化的现有路径有大量信息 在减数分裂过程中促进同源染色体的DNA修复。然而，几乎没有什么是 人类的同源染色体是如何用于修复的。中的关键决定因素之一 在HR过程中，染色体选择是突触前复合体(PSC)的组织。这项规定， 人PSC的形成和活性受&gt；45蛋白控制。然而，一个基本功能单元 PSC由有丝分裂过程中的RAD51及其相关因子(RAD54L)和RAD51、DMC1及其相关因子组成。 减数分裂相关因子(RAD54L、RAD54B、HOP2-MND1)。了解这些蛋白质是如何 在HR过程中组织成活性复合体是理解人类同源性的关键一步 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RAD51和DMC1在减数分裂过程中自分离机制的方法 队形。我们将了解DMC1是如何形成减数分裂同源搜索复合体的，并与 辅助蛋白，对齐DNA序列。我们将确定减数分裂同源复合体是如何 克服染色质。最后，我们将努力了解这两个高度相关的发动机之间的冲突 RAD54L和RAD54B蛋白可能促进人类有丝分裂过程中同源染色体的利用。在……里面 综上所述，这项研究提案的主要目标将是使用分子生物学、生物化学和单一 了解人类有丝分裂和减数分裂PSCs如何组织和促进DNA的分子方法 HR过程中的序列比对。我们从这些实验中收集的数据将被用来为 人类同源染色体选择在有丝分裂和减数分裂过程中如何发生。

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.