# Understanding Replication Stress Response in Mammalian Cells

> **NIH NIH R01** · ROCKEFELLER UNIVERSITY · 2022 · $339,000

## Abstract

Project Summary
 In each cell cycle, DNA replication machinery encounters replication fork barriers including DNA lesions,
secondary structure-forming repetitive sequences, and transcriptional machinery. Oncogenic transformation also
perturbs normal replication and results in replication fork dysfunction commonly referred to as replication stress.
Response to replication stress is an essential aspect of the DNA damage response in cells, and the
consequences of inappropriate response results in genome instability and cancer.
 We have recently identified a novel regulatory pathway that is required for the protection of stalled
replication forks and recovery from replication stress. We showed that the mammalian replisome contains a
previously unidentified and completely unstudied protein, RTF2 (Replication Termination Factor 2), which must
be removed for proper response to replication stress. We showed that RTF2 is removed from stalled forks in a
process that is dependent on the proteasomal shuttle proteins DDI1 and DDI2, which interact with RTF2 and the
proteasome. Persistence of RTF2 at stalled forks resulted in replication fork restart defects, hyperactivation of
the DNA damage signaling, accumulation of single stranded DNA, sensitivity to replication drugs including
hydroxyurea and aphidicolin, and chromosome instability. Our results establish that removal of RTF2 is
necessary for cells to manage replication stress and maintain genome integrity.
 The first goal of the proposed studies is to fully understand how RTF2 functions during DNA replication.
To this end, we will fully characterize replication without RTF2, using a conditional knockout mouse and cell
model, and identify the mechanism of how RTF2 regulates DNA replication during unperturbed conditions. The
second goal is to determine how RTF2 is itself regulated under replication stress and why it needs to be removed
from the replisome. RTF2 ubiquitination is necessary for interaction with DDI1/2, thus we will identify the
regulatory network of this ubiquitination and subsequent removal of RTF2 from the replication fork. The final goal
in this project, is to leverage the idea that the removal of proteins during DNA damage response is as equally
important as recruitment of DNA repair proteins to sites of DNA damage. Most published studies have
concentrated on proteins traveling or recruited to sites of DNA damage. However, our work on DDIs and RTF2
suggests a large component of the DNA damage response network is missing, i.e. proteins that must be removed
from the sites of damage to allow for proper DNA damage response and repair. In order to identify other proteins
removed during replication stress, we will use an approach similar to the one we used for our DDI studies and
detect proteins inappropriately enriched at stressed replication forks using a recently-developed technique,
Isolation of Proteins On Nascent DNA (iPOND). We envision that our studies will identify yet unknown regulatory...

## Key facts

- **NIH application ID:** 10491038
- **Project number:** 5R01GM140400-02
- **Recipient organization:** ROCKEFELLER UNIVERSITY
- **Principal Investigator:** Agata Smogorzewska
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $339,000
- **Award type:** 5
- **Project period:** 2021-09-20 → 2025-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10491038

## Citation

> US National Institutes of Health, RePORTER application 10491038, Understanding Replication Stress Response in Mammalian Cells (5R01GM140400-02). Retrieved via AI Analytics 2026-06-11 from https://api.ai-analytics.org/grant/nih/10491038. Licensed CC0.

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