Project Summary The genome is continuously exposed to chemical agents which modify DNA bases and damage the DNA structure. These unrepaired DNA lesions can become obstacles during replication. This results in stalling of nascent DNA synthesis and generates replication stress. Since each strand of parental DNA is replicated by distinct mechanisms (leading vs. lagging strand synthesis), DNA lesions encountered on different parental strands will generate unique forms of replication stress. Previous studies using cell culture and simple in vitro replication systems have described translesion synthesis, re-priming, and fork reversal as pathways which function to relieve replication stress. However, these approaches cannot selectively stall leading or lagging strand synthesis nor differentiate the cellular mechanisms that function on each strand. I hypothesize that leading and lagging strand replication stress are relieved by distinct mechanisms. I will differentiate between strand-specific mechanisms using the Xenopus egg extract model replication system. I have successfully constructed plasmid substrates which contain leading and lagging strand-specific lesions. My preliminary data demonstrate that lesions on different strands have different effects on the kinetics of nascent DNA synthesis, suggesting that different forms of replication stress are generated. This proposal will investigate the functional pathways which relieve strand-specific replication stress and evaluate the outcomes of each pathway. In Aim 1, I will use gel- based analyses to monitor the DNA intermediates that are formed and determine how lesions on each strand are eventually overcome. I will also use quantitative proteomics to monitor the proteins that are recruited or lost during replication of leading or lagging strand lesions. Together, these approaches will identify candidate pathways which function on different strands. In Aim 2, I will use a loss-of-function approach to determine how strand-specific pathways are controlled and monitor detrimental replication outcomes when select pathways are inactivated. This will be done by selective inhibition and immunodepletion of candidate proteins from the cell extracts. The findings of this proposal will define the specific cellular pathways which respond to specific forms of replication stress and determine how various mechanisms are employed to faithfully replicate the genome.