The ability of cells to restrict DNA replication during replication stress is critical to preserving genome integrity. We recently discovered that yeast cells lacking the Rrm3 helicase do not arrest DNA synthesis during replication stress. We found (1) that this new Rrm3 function is independent of its helicase activity and instead (2) maps to a region of the poorly characterized N-terminal tail that binds Orc5 of the origin recognition complex, and (3) that the N-terminal tail is essential for Rrm3 association with origins in the presence of replication stress, but not in unperturbed cells. We hypothesize that ORC recruits Rrm3 via its N-terminal tail to pre-replication complexes and that this association is required for inhibition of DNA synthesis during replication stress. Rrm3 is thought to use its helicase activity to ?sweep? the DNA ahead of the replisome clear to aid replication fork progression. We reasoned therefore that yeast that lacks Rrm3 makes an excellent model system for revealing the cellular response to replication fork pausing. Indeed, using quantitative proteomics we determined that the homologous recombination factor Rdh54 and the Rad5-mediated pathway for error-free lesion bypass are upregulated in the chromatin fraction of rrm3-deficient cells and that cells lacking both, Rrm3 and Rad5, accumulate DNA double strand breaks (DSBs). Moreover, the fork protection complex and polymerase are lost from the chromatin in cells lacking Rad5. Based on these findings we hypothesize that Rad5 defines a major DSB prevention mechanism that is required to overcome stalling and possibly collapse of paused forks in the rrm3? mutant. We further hypothesize that Rad5 accomplishes this by mediating PCNA polyubiquitination to regulate error-free bypass of fork blocks, such as DNA-bound proteins that accumulate on DNA in the absence of the Rrm3 sweepase activity, and (ii) by stabilizing replisome components that are required for coordinated restart. The experiments designed to test these hypotheses will (1) identify the mechanism by which Rrm3 restricts DNA synthesis during replication stress, (2) determine the mechanism by which Rrm3-Orc5 binding regulates origin association, origin activity, and DNA synthesis during replication stress and (3) define the cellular response to increased replication fork pausing. We expect that accomplishing the aims of this proposal will shed new light on fundamental mechanisms that maintain the integrity of DNA replication initiation and elongation in eukaryotic cells. We expect our findings to establish Rrm3 as a component not only of the replisome, but also of the pre-initiation complex at origins. What we learn about the role of Rrm3 in preventing replication fork blocks and about the role of Rad5 and Rdh54 in repairing these blocked forks by an error-free mechanism will also help to clarify how human cells deal with replication fork blocks and better define the role of the Rad5 ortholog HLTF in suppressing tumorigenesis.

Public Health Relevance

The causes of genome instability, which is a hallmark of most cancers, are still unclear. The proposed studies will provide mechanistic insights into the role of the DNA unwinding enzyme Rrm3 at origins of replication and in the replication stress response in budding yeast, a eukaryotic model system for studying fundamental processes of DNA replication, repair and recombination. The study will shed light on the response of eukaryotic cells to blocked replication forks, which can lead to toxic DNA breaks if unrepaired.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM132821-01A1
Application #
9998498
Study Section
Molecular Genetics A Study Section (MGA)
Program Officer
Reddy, Michael K
Project Start
2020-05-01
Project End
2024-04-30
Budget Start
2020-05-01
Budget End
2021-04-30
Support Year
1
Fiscal Year
2020
Total Cost
Indirect Cost
Name
University of South Florida
Department
Microbiology/Immun/Virology
Type
Schools of Arts and Sciences
DUNS #
069687242
City
Tampa
State
FL
Country
United States
Zip Code
33617