# Role of Pif1 family DNA helicase Rrm3 in regulating DNA synthesis during replication stress

> **NIH NIH R01** · UNIVERSITY OF SOUTH FLORIDA · 2020 · $296,950

## Abstract

PROJECT SUMMARY
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...

## Key facts

- **NIH application ID:** 9998498
- **Project number:** 1R01GM132821-01A1
- **Recipient organization:** UNIVERSITY OF SOUTH FLORIDA
- **Principal Investigator:** Kristina Schmidt
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $296,950
- **Award type:** 1
- **Project period:** 2020-05-01 → 2024-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9998498

## Citation

> US National Institutes of Health, RePORTER application 9998498, Role of Pif1 family DNA helicase Rrm3 in regulating DNA synthesis during replication stress (1R01GM132821-01A1). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/9998498. Licensed CC0.

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