SLX4 in Nuclease Recruitment

项目摘要和摘要 电离辐射和化学制剂会导致DNA链断裂，从而导致突变和 会导致癌症的染色体改变。针对DNA链的主要生物防御之一 断裂是双链断裂(DSB)修复，是一个复杂的生物通路家族。DSB的几种模式 修复可以通过DNA序列的完全恢复来进行，但其他修复会导致显著的序列变化 或者是损失。关于路径选择的生化要求，仍有许多重要的问题。 在这些更广泛的问题中，有关于征聘机制的更具体的问题 参与某些DSB修复途径但不参与其他修复途径的蛋白质。在面包酵母(S.cerevisiae)中，SLX4 蛋白质将几种内切酶中的任何一种招募到DSB位点，这些酶要么移除无关的 不同源的DNA延伸或切割DNA的四向连接(Holliday Junction)作为最后的 维修事件的步骤。SLX4征集的内切酶包括Rad1-Rad10、MUS81-Mms4和Yen1， 但是，关于选择一种内切酶而不是另一种内切酶的生化细节，在 足够的细节。越来越清楚的是，细胞周期的不同阶段在 DSB位点的内切酶访问和参与。该项目将详细说明SLX4在招聘 酿酒酵母中DSB的内切酶，关注为什么SLX4基因是招募所需要的 核酸内切酶RAD1-RAD10在细胞周期的某些阶段表达，而在其他阶段则不表达。这样做的具体目的是 建议是：1)确定不积极参与细胞分裂的细胞(处于G1期的细胞)是否需要 SLX4用于修复产物形成，2)调查Rad1-Rad10是否招募到几种特定类型的 DSB站点仅在S阶段依赖于SLX4，3)调查SLX4是否抑制了检查点信号 在细胞分裂中依赖SLX4的Rad1-Rad10在DSB修复中的募集中起作用，4)决定 RAD1-RAD10是否与MUS81-MMS4或Yen1在最后一刻的DNA修复中共存 细胞分裂中染色体分离的时刻，以及这种定位是否依赖于SLX4。 这些目标将通过各种实验技术进行研究，包括一种相对新颖的 诱导双链断裂并用荧光监测其修复的荧光显微镜方法 会聚荧光信号的成像。定量聚合酶链式反应和染色质等体外技术 免疫沉淀也将用于提供确证结果。这些实验将解决 关于招募RAD1-RAD10的遗传和生化要求的重要问题， MUS81-MMS4和Yen1到DSB站点。这三种核酸酶在包括人类在内的所有真核生物中都是保守的。 了解基因组不稳定的分子基础将有助于我们理解癌症的原因 并将对推进将人类痛苦降至最低的临床战略起到重要作用。

PROJECT SUMMARY AND ABSTRACT Ionizing radiation and chemical agents induce strand breaks in DNA, which give rise to mutations and chromosomal alterations that can cause cancer. One of the chief biological defenses against DNA strand breaks is Double-Strand Break (DSB) repair, a complicated family of biologic pathways. Some modes of DSB Repair can proceed with full restoration of the DNA sequence, but others result in significant sequence change or loss. Many important questions remain regarding biochemical requirements for pathway selection. Within these broader issues are more specific questions regarding the mechanism of recruitment of proteins that participate in some DSB repair pathways but not others. In baker's yeast (S. cerevisiae), Slx4 protein recruits any of several endonucleases to DSB sites, which either remove an extraneous nonhomologous stretch of DNA or incise a four-way junction of DNA (a Holliday Junction) as one of the last steps of the repair event. The endonucleases recruited by Slx4 include Rad1-Rad10, Mus81-Mms4 and Yen1, but the biochemical details governing the selection of one endonuclease over another are not understood in sufficient detail. It is becoming increasingly clear that the phase of the cell cycle plays a critical role in endonuclease access and engagement of the DSB site. This project will detail the role of Slx4 in recruitment of endonucleases to DSBs in the yeast S. cerevisiae, focusing on why the SLX4 gene is needed for recruitment of endonuclease Rad1-Rad10 in some phases of the cell cycle but not others. The specific aims of this proposal are to: 1) determine whether cells that are not actively engaged in cell division (those in G1) require SLX4 for repair product formation, 2) investigate whether Rad1-Rad10 recruitment to several specific types of DSB sites depends on SLX4 only in S phase, 3) investigate whether checkpoint signal dampening by Slx4 plays a role in SLX4-dependent recruitment of Rad1-Rad10 in DSB repair in dividing cells and, 4) determine whether Rad1-Rad10 colocalizes with Mus81-Mms4 or Yen1 in last-minute DNA repair during the final moments of chromosome separation in cell division and if such localization is SLX4-dependent. These aims will be investigated with a variety of experimental techniques, including a relatively novel fluorescence microscopy approach in which DSBs will be induced and their repair monitored by fluorescence imaging of convergent fluorescent signals. In vitro techniques such as quantitative PCR and Chromatin Immunoprecipitation will also be used to provide corroborating results. These experiments will address important questions regarding the genetic and biochemical requirements for recruitment of Rad1-Rad10, Mus81-Mms4 and Yen1 to DSB sites. All three nucleases are conserved in all eukaryotes including humans. Understanding the molecular basis for genome instability will inform our understanding of the causes of cancer and aging, and will be important for advancing clinical strategies to minimize human suffering.