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Research Abstracts Online
January 2010 - March 2011

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University of Minnesota Twin Cities
College of Biological Sciences
Medical School
Department of Biochemistry, Molecular Biology, and Biophysics

PI: Anja-Katrin Bielinsky

Investigation of Replication Fork Progression in CDC9 (DNA Ligase I) Mutants, Which Have Okazaki Fragment Maturation Defects

DNA ligase I, encoded by CDC9 in budding yeast, catalyzes the ligation of newly synthesized DNA on the lagging strand known as Okazaki fragments. An individual with a DNA ligase I deficiency exhibited growth retardation, sunlight sensitivity, and severe immunosuppression, likely due to accumulation of DNA damage. However, not much is known about the DNA damage response (DDR) in DNA ligase I-deficient cells. Using budding yeast as a model system, these researchers recently reported that DNA ligase I is required for S phase progression. Moreover, CDC9 mutants progress through S phase significantly more slowly than wild-type cells. The delay in S phase in these mutants requires the S-phase checkpoint kinase, Rad53 in an Mrc1- and Rad9-dependent manner. Most importantly, the researchers discovered PCNA ubiquitination occurring at a novel site, K107, which is a prerequisite for Rad53 activation. They hypothesize that the delay in S phase progression is likely due to the inhibition of late-firing origins while maintaining a few early-firing replication forks to replicate the whole genome. To test this hypothesis, the researchers have constructed CDC9 mutants that are capable of incorporating BrdU into nascent DNA. They will perform genome-wide analysis of DNA synthesis by BrdU immunoprecipitation on tiling microarrays (BrdU-IP-chip) in these CDC9 mutants. This will allow them to identify which replication origins are activated and monitor replication fork progression in DNA ligase I-deficient cells. They will also examine the role of PCNA ubiquitination at K107 on de novo DNA synthesis. These studies will provide insight into the mechanism of how cells maintain genome stability when lagging strand DNA synthesis is compromised.

Group Member

Hai Dang Nguyen, Graduate Student