Meiotic recombination in budding yeast

有性繁殖的基础是产生配子的能力，如精子和卵子 人类，每条染色体只有一个拷贝。然后，受精导致两种 单倍体配子形成二倍体有机体。对于像发芽酵母和人类这样的生物来说，这是一种 这是一项艰巨的任务，因为分别有16对和23对染色体必须进行适当的分类 每一个配子。减数分裂是一种特殊的细胞分裂，它通过以下方式将染色体数量减半 一轮DNA复制后两轮染色体分离。未能妥善处理 在减数分裂过程中分离染色体会产生染色体不平衡的配子，导致不育。 以及出生缺陷，如21三体(唐氏综合症)。 减数分裂的一个关键部分是第一次减数分裂，在那里同源染色体被分离。 至主轴的相反两极。交叉是由DNA的相互交换产生的 同系物。交叉，结合姐妹染色单体凝聚力，在物理上连接同源基因，因此它们 可以在第一次减数分裂时对齐并适当分离。双链修复产生的交叉 故意引入染色体以启动重组的断裂。因为没有修好的两倍 链断裂是致命的，减数分裂重组是一个高度调控的过程，确保每一对 同源基因至少有一次交叉，所有双链断裂都在第一次减数分裂之前修复 组织。 直接研究哺乳动物的减数分裂是困难的，因为它很难获得生殖细胞，而这些细胞 二倍体，这使得寻找隐性突变具有挑战性。萌芽酵母，酿酒酵母，有 是研究减数分裂的一个很好的模型系统，因为复杂的遗传，生化， 可用的分子和细胞生物学工具。我的研究重点一直是识别基因 减数分裂重组所需，并确定这些基因发挥作用的分子机制 和/或受到监管。特别是，我的实验室开发了新的方法来研究磷酸化如何 调节减数分裂过程中的重组，重点是减数分裂特异的激酶、MEK1和 保守的细胞周期蛋白激酶，CDC7-Dbf4。 尽管我们对减数分裂重组的理解已经取得了巨大的进展，但仍有 仍然是需要填补的关键差距。例如，有许多基因有助于 减数分裂双链断裂修复有待发现。在接下来的五年里，我的实验室计划 研究两个以前未知的在减数分裂中起作用的基因：SEN1和RRM3。 Sen1是一种解旋酶，在有丝分裂的细胞中解开被称为R环的RNA/DNA杂合体，是必不可少的 终生如此。它的哺乳动物同源基因senataxin是减数分裂所必需的。RRM3是保守的Pif1的成员 DNA解旋酶家族，因其在DNA复制中的作用而广为人知。此外，我们还发现了一个 另外两个DNA解旋酶SGs1和srs2在调节 交叉编队。 许多生物如酵母和哺乳动物的减数分裂需要两种重组酶，RAD51，它是 对有丝分裂重组和减数分裂特异的Dmc1至关重要，Dmc1介导了大部分同源基因 重组。一个悬而未决的问题是为什么需要两个重组酶。我的实验室和其他人的工作 已经提出，Dmc1已经进化为更好地处理可能由以下原因引起的不匹配的基对 同源链间侵袭，因为同源DNA高度相似，但不一定相同 序列。我的实验室已经开发出一种活体方法来测试这一有趣的假设。支持的工作 这笔赠款将对我们理解减数分裂做出重要贡献，知识可能 最终适用于人类预防/治疗不孕不育和出生缺陷。

Fundamental to sexual reproduction is the ability to make gametes, such as sperm and eggs in humans, which contain only one copy of each chromosome. Fertilization then results in the fusion of two haploid gametes to create a diploid organism. For organisms such as budding yeast and humans, this is a daunting task, as there are 16 and 23 pairs of chromosomes, respectively, that must be properly sorted into each gamete. Meiosis is the specialized cell division that divides the chromosome number in half by having one round of DNA replication followed by two rounds of chromosome segregation. Failure to properly segregate chromosomes during meiosis produces chromosomally imbalanced gametes, resulting in infertility and birth defects such as Trisomy 21 (Down Syndrome). A critical part of meiosis is the first meiotic division, where homologous chromosomes are segregated to opposite poles of the spindle. Crossovers are created by the reciprocal exchange of DNA between homologs. Crossovers, in combination with sister chromatid cohesion, physically connect homologs so they can align and properly segregate at the first meiotic division. Crossovers result from the repair of double strand breaks that are deliberately introduced into chromosomes to initiate recombination. Because unrepaired double strand breaks are lethal, meiotic recombination is a highly regulated process that ensures that every pair of homologs receives at least one crossover and that all double strand breaks are repaired before the first meiotic division. Studying meiosis directly in mammals is difficult as it is hard to access germ cells and the cells are diploid, making it challenging to find recessive mutations. The budding yeast, Saccharomyces cerevisiae, has been an excellent model system for studying meiosis because of the sophisticated genetic, biochemical, molecular and cell biological tools that are available. The focus of my research has been on identifying genes required for meiotic recombination and defining the molecular mechanisms by which these genes function and/or are regulated. In particular, my lab has developed novel approaches for studying how phosphorylation regulates recombination during meiosis, with an emphasis on the meiosis-specific kinase, Mek1 and the conserved cell cycle kinase, Cdc7-Dbf4. Although immense progress has been made in our understanding of meiotic recombination, there are still critical gaps that need to be filled. For example, there are many genes that contribute to the fidelity of meiotic double strand break repair which remain to be discovered. Over the next five years, my lab plans to study two genes we have identified that were previously unknown to play a role in meiosis: SEN1 and RRM3. Sen1 is a helicase that unwinds RNA/DNA hybrids called R-loops in mitotically dividing cells and is essential for life. Its mammalian ortholog, Senataxin, is required for meiosis. Rrm3 is a member of the conserved Pif1 DNA helicase family that is well known for its role in DNA replication. In addition, we have discovered a potentially novel role for two other DNA helicases, Sgs1 and Srs2, working together in the regulation of crossover formation. Meiosis in many organisms such as yeast and mammals requires two recombinases, Rad51, which is essential for mitotic recombination and the meiosis-specific Dmc1, which mediates the bulk of interhomolog recombination. An outstanding question is why two recombinases are necessary. Work from my lab and others has suggested that Dmc1 has evolved to better handle the mismatched basepairs that can arise by interhomolog strand invasion because homologs have highly similar, but not necessarily identical DNA sequences. My lab has developed an in vivo approach to test this interesting hypothesis. The work supported by this grant will make an important contribution to our understanding of meiosis, knowledge which may ultimately be applicable in humans for preventing/treating infertility and birth defects.