Control and impact of meiotic DNA resection on recombination and genome stability

同源重组（homembryonic recombination，HR）是一个被广泛研究的过程，因为它对生育力、生物多样性和基因组稳定性至关重要。在动物、植物和真菌（如芽殖酵母模式生物酿酒酵母）中，每个细胞都含有两个染色体拷贝：一个从母亲遗传，一个从父亲遗传。在被称为“减数分裂”的特殊细胞分裂过程中，HR发生在这些染色体对之间，产生配子（卵子和精子），每个配子都含有亲本基因的独特组合。减数分裂HR由分布在所有亲本染色体上的许多DNA断裂启动。减数分裂细胞使用HR来修复这些DNA断裂，以产生所谓的“交叉”（CO）和“非交叉”（NCO），这两者对单个配子的遗传变异都很重要，因此对生物多样性和作物育种至关重要。CO对于减数分裂期间的准确染色体分离也是必不可少的，通过防止非整倍性（一种基因组不稳定性，其特征在于配子具有不正确的染色体数目），这是不孕症和遗传疾病（如唐氏综合症）的常见原因。HR对修复自发产生或由环境诱导的DNA错误也至关重要，HR缺陷可赋予遗传不稳定性，从而引发癌症发展。HR的一个中心步骤是通过各种酶降解DNA双螺旋的两条DNA链中的一条来处理DNA断裂，产生单链DNA（ssDNA）的短区域。这个过程被称为“切除”。这些ssDNA区域被进化上保守的修复酶（称为RecA，Rad51和Dmc 1）用于搜索基因组中其他地方存在的类似DNA序列。这些相似的序列被用作复制的模板，从而修复DNA断裂。切除的起始步骤（接合并提交待由HR修复的DNA末端）被很好地表征，但是关闭切除的机制以及切除长度、基因组稳定性和遗传变异之间的联系知之甚少。尽管有许多令人信服的迹象表明切除长度将影响基因组稳定性和遗传变异。例如，在恶性肿瘤的全基因组测序中鉴定的突变簇与在切除过程中出现的ssDNA上发生的DNA损伤有关。此外，我们未发表的工作表明，解除管制的切除是一个主要的危险因素与减数分裂过程中异常重组，产生不平衡的染色体重排，降低fertility.In本提案中，我们将使用先进的全基因组技术在芽殖酵母模式生物，以检查如何在基因组中的所有位置的DNA切除终止调节。DNA断裂激活检查点途径，延迟细胞分裂，直到DNA修复。我们将研究切除是如何通过检查点途径调节的，以及染色体的结构特征如何影响整个基因组不同位置的切除过程。重要的是，我们将使用先进的遗传学方法来研究切除程度的变化如何影响HR的机制，特别是对基因组稳定性和遗传变异的影响。我们的工作，“发现科学”，对于理解切除以及更广泛的HR是否，如何以及为什么控制生育力，生物多样性，基因组稳定性和癌症具有根本的重要性。

Homologous recombination (HR) is a widely studied process because of its fundamental importance for fertility, biodiversity, and genome stability.In animals, plants and fungi (such as the budding yeast model organism Saccharomyces cerevisiae) every cell contains two copies of each chromosome: one inherited from the mother and one from the father. During the specialised cell division called "meiosis", HR occurs between these chromosome pairs producing gametes (eggs and sperm) that each contains a unique combination of the parental genes. Meiotic HR is initiated by numerous DNA breaks spread across all the parental chromosomes. Meiotic cells use HR to repair these DNA breaks to create so-called "crossovers" (COs) and "non-crossovers" (NCOs), both of which are important for genetic variation in individual gametes, and therefore essential for biodiversity and crop breeding. COs are also essential for accurate chromosome segregation during meiosis, by preventing aneuploidy (a type of genome instability characterised by gametes with an incorrect number of chromosomes), which is a common cause of infertility and genetic disorders such as Down Syndrome. HR is also critical to repair errors in the DNA arising spontaneously or induced by the environment, and HR defects can confer genetic instability, which can then trigger cancer development.A central step of HR is the processing of DNA breaks by various enzymes that degrade one of the two DNA strands of the DNA double helix, generating short regions of single-stranded DNA (ssDNA). This process is referred to as "resection". These ssDNA regions are used by evolutionarily conserved repair enzymes (called RecA, Rad51 and Dmc1) to search for similar DNA sequences present elsewhere in the genome. These similar sequences are used as templates from which to copy, and thereby repair, the DNA breaks.The initiation step of resection (engaging and commiting the DNA end to be repaired by HR) is well characterised, but the mechanisms that switch off resection, and the link between resection length, genome stability and genetic variation is poorly understood. This is despite a number of compelling indications that resection length will impact genome stability and genetic variation. For example, clusters of mutations identified in whole-genome sequencing of malignant tumors are linked to DNA damage occurring on the ssDNA that arises during resection. Furthermore, our unpublished work indicates that deregulated resection is one of the major risk factors associated with aberrant recombination during meiosis, generating imbalanced chromosome rearrangements that reduce fertility.In this proposal we will use advanced whole-genome technologies in the budding yeast model organism to examine how DNA resection termination is regulated at all locations in the genome. DNA breaks activate checkpoint pathways that delay cell division until the DNA is repaired. We will study how resection is regulated by the checkpoint pathway, and how structural features of the chromosome affect the resection process at different locations across the genome. Importantly, we will use advanced genetic methods to examine how changes in the extent of resection influence the mechanism of HR, with particular interest in the impact on genome stability and genetic variation.Our work, "discovery science", will be of fundamental importance to understand if, how and why resection, and more broadly HR, control fertility, biodiversity, genome stability and cancer.