Identifying genetic determinants ensuring correct DNA damage repair pathway choice

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 PhD
project 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: PMC3728922
2. 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 for
normal meiotic recombination partner choice. Genetics 153(2): 607-620. PMID: 10511543, PMCID: PMC1460798