# Investigation of strand-specific DNA replication stress response mechanisms.

> **NIH NIH F32** · VANDERBILT UNIVERSITY · 2022 · $67,174

## Abstract

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.

## Key facts

- **NIH application ID:** 10536820
- **Project number:** 1F32GM148024-01
- **Recipient organization:** VANDERBILT UNIVERSITY
- **Principal Investigator:** Matthew Cranford
- **Activity code:** F32 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $67,174
- **Award type:** 1
- **Project period:** 2022-09-01 → 2023-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10536820

## Citation

> US National Institutes of Health, RePORTER application 10536820, Investigation of strand-specific DNA replication stress response mechanisms. (1F32GM148024-01). Retrieved via AI Analytics 2026-05-26 from https://api.ai-analytics.org/grant/nih/10536820. Licensed CC0.

---

*[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*