Research Abstracts Online
January - December 2011
University of Minnesota Twin Cities
College of Biological Sciences
of Biochemistry, Molecular Biology, and Biophysics
PI: Anja-Katrin Bielinsky
Accumulation of Nicked DNA Induces PCNA Ubiquitination, Homologous Recombination, and Single Strand Annealing
DNA ligase I is an essential protein involved in the ligation of Okazaki fragments. These researchers have previously shown that cells trigger a novel ubiquitination pathway that targets proliferating cell nuclear antigen (PCNA) at lysine (K) 107 in Saccharomyces cerevisiae cdc9 (DNA ligase I) mutants. PCNA ubiquitination at K107 is crucial for the activation of the S phase checkpoint kinase Rad53 in cdc9 mutants (Das-Bradoo et al, 2010b). To determine that PCNA ubiquitination in DNA ligase I-deficient cells is due to deficient DNA ligase activity, the researchers complemented cdc9-1 cells with either wild-type or mutant Cdc9. While expression of a wild-type Cdc9 fully reversed PCNA ubiquitination in cdc9-1 cells, expression of two inactive Cdc9 mutants, K419A and K598A did not. These mutants resulted in the accumulation of “clean” and “dirty” nicks, respectively, demonstrating that both types of nicks are sufficient to induce PCNA mono- and poly-ubiquitination.
To better understand how cells cope with the accumulation of nicks during Okazaki fragment maturation, and what the molecular function of PCNA ubiquitination might be, the researchers utilized cdc9-1 in a genome-wide synthetic lethality screen. Consistent with previous reports, genes in the RAD52 epistasis group were synthetically lethal with cdc9-1, illustrating a crucial role of double strand break (DSB) repair by homologous recombination in cdc9-1 cells. The researchers also identified genes in the single strand annealing (SSA) pathway (RAD59, RAD1 and RAD10) that may also aid in the resolution of double stranded breaks. Surprisingly, however, deletion of SLX4, a crucial component of the SSA pathway in response to DSBs, did not affect cdc9-1 mitotic growth. These results suggest that components of the SSA pathway in cdc9-1 cells function independently of the DSB repair pathway and may be required to resolve the accumulation of DNA nicks. The researchers plan a template switch model by which Okazaki fragments anneal to the nascent leading strand via single strand annealing for continuous DNA synthesis, thus bypassing the requirement for DNA ligase I.
Hai Dang Nguyen, Graduate Student