Molecular Mechanisms of Human Homologous Recombination

总结/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有助于大多数癌症的发展。DNA双链断裂 是一种危险的DNA损伤类型，可导致存储在细胞内的序列信息迅速丢失。 基因组同源重组（HR）是DSB修复的主要途径之一， 定位与基因组中其他地方的受损DNA序列匹配的未受损DNA序列。 然后，同源序列可用于恢复丢失的DNA序列信息。在正常 在有丝分裂生长中，HR优先使用储存在姐妹染色单体中的序列信息修复DSB。 帮助维持基因间的等位变异并防止遗传交换的不平衡 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 优选的DNA修复底物。有大量的信息，现有的途径，已经演变， S.在减数分裂期间，酿酒酵母促进同源染色体的DNA修复。然而， 我们知道同源染色体是如何用于人类的修复的。其中一个关键的决定因素， 在HR期间的染色体选择是突触前复合体（PSC）的组织。条例， 人PSC的形成和活性受>45种蛋白质控制。然而， PSC由有丝分裂过程中的RAD 51和相关因子（RAD 54 L）组成，以及RAD 51、DMC 1和它们的相互作用。 RAD 54 L、RAD 54 B、HOP 2-MND 1等相关因子。了解这些蛋白质 在HR期间组织成活性复合物是理解人类同源 染色体用于HR。在我们的研究过程中，我们将使用生物化学和单分子 了解减数分裂PSC过程中RAD 51和DMC 1自我分离机制的方法 阵我们将了解DMC 1如何形成减数分裂同源性搜索复合物，并与 辅助蛋白，比对DNA序列。我们将确定如何减数分裂同源搜索复合物 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RAD 54 L和RAD 54 B可能在人类有丝分裂HR期间促进同源染色体的使用。 总之，这项研究计划的主要目标将是利用分子生物学，生物化学和单 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 我们从这些实验中收集的数据将用于建立一个模型， 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。

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.