Identifying genetic determinants ensuring correct DNA damage repair pathway choice

识别遗传决定因素，确保选择正确的 DNA 损伤修复途径

• 批准号: 2432830
• 金额: --
• 依托单位: University of Nottingham
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2020
• 资助国家: 英国
• 起止时间: 2020 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2432830
• 关键词: Identifying; genetic; determinants; ensuring; correct

DNA double-strand breaks (DSBs), where both strands of the DNA helix are broken at the same locus, are the most dangerous lesion to occur to the genome. DSBs must be repaired correctly and in a timely manner to ensure genomic stability. One main pathway for DSB repair is homologous recombination, where a similar DNA sequence, usually the sister chromatid at the same locus, is used as a template for repair. However, when homologous recombination repair takes place from a similar DNA sequence at an incorrect locus, ectopic recombination occurs leading to the formation of chromosomal aberrations, manifested as mutations, deletions, additions and translocations.Investigating the mechanisms that inhibit or promote ectopic recombination using the budding yeast Saccharomyces cerevisiae is the aim of this PhD project and is broken down into three elements, described below.Our current knowledge of ectopic recombination inhibitors are DNA repair and DNA damage checkpoint proteins. These proteins appear to regulate DSB repair pathways to prevent ectopic recombination. Intriguingly our results so far indicate that mutation of individual components of the same DNA damage checkpoint lead to different levels of ectopic recombinants.The first element of this PhD project will be to define the types and levels of ectopic recombinants that form in the different checkpoint mutants. This will be undertaken using established Southern and western blot assays, use of a novel fluorescent reporter tool and development of a CRISPR/Cas9 sequencing technique.Our current data suggests that ectopic recombinants can form by different DNA repair mechanisms. To this end, the second element of this PhD project will be to evaluate and characterise the types of ectopic recombinants formed in mutants defective in specific DNA repair pathways.Our understanding of ectopic recombination has so far focused on strategic and directed investigations, given previously characterised roles of proteins. However, this strategy removes the ability to find novel inhibitors of ectopic recombination. To this end, the final element of this PhDproject will to be develop the use of CRISPR/Cas9 targeted mutation in reporter strains. Once established, the student will construct a CRISPR library and undertake a screen allowing for enrichment of cells carrying ectopic recombinants. Following sequencing to identify the gene mutated, further characterisation of the mutant will be undertaken to define the mechanism for ectopic recombination in this background. Observations made throughout this project will be have the potential to be developed in additional model organism systems.References to learn more:1. Gray, S., Allison, R.M., Garcia, V., Goldman, A.S.H., Neale, M.J., (2013) Positive regulation of meiotic DNA double-strand break formation by activation of the DNA damage checkpoint kinase Mec1(ATR). Open Biol 3: 130019. doi: 10.1098/rsob.130019 PMID: 23902647, PMCID: PMC37289222. Grushcow, J. M., Holzen, T. M., Park, K. J., Weinert, T., Lichten, M., Bishop, D. K. (1999). Saccharomyces cerevisiae checkpoint genes MEC1, RAD17 and RAD24 are required fornormal meiotic recombination partner choice. Genetics 153(2): 607-620. PMID: 10511543, PMCID: PMC1460798

DNA 双链断裂 (DSB)，即 DNA 螺旋的两条链在同一位点断裂，是基因组发生的最危险的损伤。 DSB 必须及时正确修复，以确保基因组稳定性。 DSB 修复的一个主要途径是同源重组，其中相似的 DNA 序列（通常是同一位点的姐妹染色单体）被用作修复模板。然而，当相似的 DNA 序列在不正确的位点发生同源重组修复时，就会发生异位重组，导致染色体畸变的形成，表现为突变、缺失、添加和易位。利用芽殖酵母酿酒酵母研究抑制或促进异位重组的机制是本博士项目的目的，也是 分为三个要素，如下所述。我们目前对异位重组抑制剂的了解是 DNA 修复和 DNA 损伤检查点蛋白。这些蛋白质似乎可以调节 DSB 修复途径以防止异位重组。有趣的是，我们迄今为止的结果表明，同一DNA损伤检查点的各个成分的突变会导致不同水平的异位重组体。这个博士项目的第一个要素将是定义在不同检查点突变体中形成的异位重组体的类型和水平。这将使用已建立的 Southern 和 Western blot 测定、新型荧光报告工具的使用以及 CRISPR/Cas9 测序技术的开发来进行。我们目前的数据表明，异位重组体可以通过不同的 DNA 修复机制形成。为此，该博士项目的第二个要素将是评估和表征在特定 DNA 修复途径缺陷的突变体中形成的异位重组的类型。考虑到先前表征的蛋白质作用，我们对异位重组的理解迄今为止集中在战略和定向研究上。然而，这种策略消除了寻找新型异位重组抑制剂的能力。为此，该博士项目的最后一个部分将是开发 CRISPR/Cas9 靶向突变在报告菌株中的应用。一旦建立，学生将构建 CRISPR 文库并进行筛选，以富集携带异位重组体的细胞。通过测序鉴定突变基因后，将对突变体进行进一步表征，以确定在此背景下异位重组的机制。在整个项目中进行的观察将有可能在其他模型生物系统中得到发展。了解更多信息的参考文献：1。 Gray, S.、Allison, R.M.、Garcia, V.、Goldman, A.S.H.、Neale, M.J., (2013) 通过激活 DNA 损伤检查点激酶 Mec1 (ATR) 对减数分裂 DNA 双链断裂形成的正调控。开放生物 3：130019。doi：10.1098/rsob.130019 PMID：23902647，PMCID：PMC37289222。 Grushcow, J. M.、Holzen, T. M.、Park, K. J.、Weinert, T.、Lichten, M.、Bishop, D.K. (1999)。酿酒酵母检查点基因 MEC1、RAD17 和 RAD24 是正常减数分裂重组伴侣选择所必需的。遗传学 153(2)：607-620。 PMID：10511543，PMCID：PMC1460798