Identifying genetic determinants ensuring correct DNA damage repair pathway choice

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病变。DSB必须正确和及时地修复，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，通常是同一基因座的姐妹染色单体，被用作修复的模板。然而，当同源重组修复发生在一个不正确的基因座上的相似DNA序列时，异位重组发生，导致染色体畸变的形成，表现为突变，缺失，添加和易位。研究使用芽殖酵母酿酒酵母抑制或促进异位重组的机制是这个博士项目的目标，并分为三个元素，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。这些蛋白质似乎调节DSB修复途径以防止异位重组。有趣的是，到目前为止，我们的研究结果表明，同一DNA损伤检查点的单个组分的突变导致不同水平的异位重组体。这个博士项目的第一个元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印迹分析，使用新的荧光报告工具和CRISPR/Cas9测序技术的发展进行。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，这个博士项目的第二个要素将是评估和鉴定在特定DNA修复途径缺陷的突变体中形成的异位重组体的类型。我们对异位重组的理解迄今为止集中在战略和定向调查上，给出了以前表征的蛋白质的作用。然而，这种策略消除了发现异位重组的新抑制剂的能力。为此，该博士项目的最后一项内容将是开发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。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。在整个项目中所做的观察将有可能在其他模式生物系统中得到发展。Gray，S.，艾莉森，R. M.，加西亚，高盛，A.S.H.，尼尔，M. J.，（2013）通过激活DNA损伤检查点激酶Mec 1（ATR）对减数分裂DNA双链断裂形成的正调控。Open Biol 3：130019. doi：10.1098/rsob.130019 PMID：23902647，PMCID：PMC37289222。Grushcow，J.M.，Holzen，T. M.，帕克，K. J.，Weinert，T.，Lichten，M.，Bishop，D. K.（1999年）。酿酒酵母检查点基因MEC 1、RAD 17和RAD 24是正常减数分裂重组伴侣选择所必需的。Genetics 153（2）：607-620. PMID：10511543，PMCID：PMC1460798

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