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Genetic determinants of genome stability and crossover distribution in the nematode Caenorhabditis elegans Chung, George Chih Hsuen


Guanine-rich DNA sequences can form secondary structures in vitro. In vivo, helicases are responsible for undoing these structures during DNA replication. In the absence of helicase function, these structures may be unrepaired and undergo mutagenic end-joining processes that generate DNA sequence deletions. In Caenrhabditis elegans, a majority of these guanine-rich structures are resolved by the helicase DOG-1. This report presents genetic evidence that a related helicase, CHL-1, acts as a backup mechanism through which guanine-rich structures may be bypassed or resolved in the absence of DOG-1 function. In the absence of CHL-1 function, DNA double-strand breaks accumulate and are repaired inefficiently in the meiotic germline. In an attempt to generate a translational fusion tag for chl-1 in order to characterize the localization of the CHL-1 helicase in the germline, I generated an unexpected knock-out allele of chl-1 using the emerging CRISPR-Cas9 technology adapted for C. elegans. Taking lessons learned from this approach, I used the CRISPR-Cas9 reagents to generate new alleles of y18h1a.7, a candidate coding sequence for the rec-1 gene. Classical genetic studies showed that a mutation in rec-1 altered the meiotic recombination pattern across the autosomes in C. elegans. The new alleles of y18h1a.7 enabled complementation testing and confirmed that the coding sequence for rec-1 is y18h1a.7 on Chromosome I. Through a synteny analysis of six other Caenorhabditis genomes in the region surrounding rec-1, I identified putative rec-1 orthologues which have limited sequence similarity to rec-1. Furthermore, the similarity of these rec-1 orthologues to C. elegans him-5 suggests that him-5 is a paralogue of rec-1. By genetic analysis, I show that rec-1 and him-5 function in partial redundancy to initiate meiotic recombination events. Thus, rec-1 and him-5 define a new class of early meiosis genes in several Caenorhabditis species. In summary, this thesis brings together the findings on chl-1 and rec-1 by highlighting their respective roles in the meiotic germline of Caneorhabditis elegans.

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