Investigating the role of DNA repair enzymes in DNA replication and recombination (735.3)

Abstract

Hypothesis: A mutant DNA repair protein (DSS1) will be unable to facilitate Homologous Recombination Repair (HRR) within osteosarcoma cell lines. Also depletions in the DNA repair enzyme RAD51AP1 will hinder the cells ability to rescue stalled replication forks and repair replication associated Double Strand Breaks (DSB), along with causing a S‐phase check point defect in cells. In this study DNA damage was induced via exogenous and endogenous stresses, to test the fitness of cells. To gain insight into the role DNA repair proteins play after DNA damage, mutant osteosarcoma cell lines were exposed to high intensity X‐rays. The ability for cells to faithfully repair double strand breaks was assessed by fluorescent activated cell sorting and immunocytochemistry. The levels of S phase check point activation by the phosphorylation of check point kinase1 was assessed by infrared Western blot analysis. We also depleted the DNA interacting enzymes and studied the effects on DNA damage, DNA replication, and the cell cycle. It was concluded that human RAD51AP1 expressed in chicken DT40 cells caused a checkpoint defect, leading to the cells inability to arrest after damage. This phenotype has been recently validated in human cell lines. The overall goal of this research was to determine the roles DSS1 and RAD51AP1 play in DNA repair and the interaction with other recombination mediators and effectors.Grant Funding Source: NSF ATE DUE 1205059