Genomic integrity is of vital importance to the cell, as the introduction of mutations in DNA can lead to cancer, aging, and other human diseases. When the replication fork encounters a DNA damage lesion, the fork stalls, which can lead to a collapsed fork and highly mutagenic double-stranded break. Initiation of damage bypass pathways prevents this genomic instability and allows for replication to complete in a timely manner, leaving the damaged template DNA behind for later repair. The pathways comprising damage bypass are known as DNA damage tolerance (DDT) or post-replication repair (PRR). DDT contains at least two branches - translesion synthesis (TLS) and template switching (TS). TS uses the newly replicated sister chromatid as a template for replication past the DNA lesion. Despite the importance of TS to maintaining genomic integrity, little is known about its regulation. Increased understanding of this regulation is critical to learning how the cell can minimize the incidence of mutations following environmental stress. SHPRH is hypothesized to mediate TS once the replication fork encounters lesions generated by oxidative stress or alkylating agents. Experiments in yeast have suggested that TS primarily occurs in S phase, with a possible role in gap filling in G2. Unexpectedly, I found that deletion of SHPRH led to a prolonged G2 phase that increased further with alkylation damage, with no effect on S phase progression, suggesting that SHPRH plays a role after the replication fork has passed the lesion. I have also found that SHPRH-null cells display higher amounts of 53BP1, a DNA damage response protein that inhibits homologous recombination when bound to its interacting partner, Rif1. Also, our preliminary data suggest that SHPRH has sumo-targeted ubiquitin ligase (STUbL) activity, which has been shown to regulate protein localization. These observations lead me to hypothesize that SHPRH promotes the removal of 53BP1/Rif1 complexes from gaps via STUbL activity to allow for template switching. I will identify gaps in wild type and SHPRH-null cells and analyze the contribution of alkylation damage to gap formation/maintenance. I will also identify gap-associated proteins that may play a role in preventing or promoting gap filling, including 53BP1/Rif1, and analyze the interactions of these proteins with SHPRH. Additionally, I will measure TS directly in the presence and absence of SHPRH and determine whether SHPRH promotes TS. To determine the mechanism of its activity, I will investigate the ability of SHPRH to perform STUbL activity to rescue G2 arrest and gap filling. To this end, I will complement SHPRH-null cells with various domain mutants of SHPRH to ascertain which domains are required for SHPRH to promote G2 progression and gap filling. Finally, I plan to identify proteins that SHPRH regulates through STUbL activity following alkylation damage. These results will help us further understand the mechanism by which SHPRH promotes TS to ensure complete replication of DNA following damage by alkylating agents.

Public Health Relevance

Genomic integrity is of vital importance to the cell, as the introduction of mutations in DNA can lead to cancer, aging, and other human diseases. Upon DNA damage by alkylating agents, the protein SHPRH promotes DNA damage tolerance pathways, which allow the cell to finish replication efficiently and repair the damaged DNA at a later time. The proposed studies will provide molecular insights into how SHPRH maintains genomic stability after DNA is damaged by alkylating agents.

Agency
National Institute of Health (NIH)
Institute
National Institute of Environmental Health Sciences (NIEHS)
Type
Postdoctoral Individual National Research Service Award (F32)
Project #
1F32ES024631-01
Application #
8782160
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Shaughnessy, Daniel
Project Start
2014-09-01
Project End
2016-08-31
Budget Start
2014-09-01
Budget End
2015-08-31
Support Year
1
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Stanford University
Department
Biology
Type
Schools of Medicine
DUNS #
City
Stanford
State
CA
Country
United States
Zip Code
94304