Investigating the Role of DDK in Replication Associated Recombination

Joseph, Chinnu Rose (2021). Investigating the Role of DDK in Replication Associated Recombination. PhD thesis The Open University.

DOI: https://doi.org/10.21954/ou.ro.0001242b

Abstract

Dbf4-dependent kinase (DDK) is an essential Serine/Threonine protein kinase well-known for its function in DNA replication initiation. DDK also functions in various other cellular process including DNA repair mechanisms, such as mutagenesis. However, DDK roles in DNA damage tolerance remain elusive.

Using S. cerevisiae as model system, here we investigated DDK roles in replication associated recombination, a process known to be induced by DNA damage and important for error-free replication. Using different mutant alleles of DDK and conditions that do not affect its function in origin firing, we found that DDK dysfunction reduces the levels of recombination intermediates forming proximal to replication forks. The effect of DDK mutations in recombination was overlapping with the window of PCNA polyubiquitylation and largely epistatic with mms2 or rad18 mutations, affecting (poly)ubiquitylation of the replication processivity factor PCNA and error-free template switching. Moreover, the effect of DDK manifested also in mutant backgrounds in which a salvage pathway of recombination independent of PCNA poly-ubiquitylation operates. DDK mutants not only affect the sister chromatid junctions but also the inter-homolog junctions that rely on recombination events to the homologous chromosomes. Notably, we found that certain DDK mutants were specifically contributing to template switching, and not to the salvage pathway, and that the recombination function of DDK is separable from its roles in origin firing.

By ChIP quantitative analysis, we uncovered that the recombination defect of DDK mutants correlates with decreased local recruitment of Rad51 to the damaged forks, potentially caused by stabilization of the Srs2 anti-recombinase. Importantly, we also found that two different ddk mutants with shared defects in template switching but differential effects on the salvage pathway and checkpoint activation accumulate long single-stranded DNA (ssDNA) gaps at the replication fork junction and broken forks. The recombination defect of ddk mutants was not rescued by downregulation of the Mus81-Mms4 nuclease, differently from what observed in checkpoint mutants, nor by artificial local restoration of cohesion. Taken together, our results uncover roles for DDK in supporting replication-associated recombination by regulating Rad51 availability at sites of perturbed replication.

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