# Understanding the regulatory mechanisms for Replication Termination Factor 2 (RTF2) removal and function during DNA replication

> **NIH NIH F32** · ROCKEFELLER UNIVERSITY · 2021 · $66,390

## Abstract

Project Summary/ Abstract
 DNA replication is a highly regulated process that occurs in the physical arena of chromatin. The DNA
replication machinery organizing this process, the replisome, confronts many obstacles, both endogenous and
exogenous, that threaten the accurate and efficient duplication of the genome. Cells have multiple mechanisms
to respond to these challenges and bypass lesions. Still, DNA replication stress has emerged as a potent source
of endogenous damage driving genome instability and cancer. Our laboratory has identified a novel replication
stress response axis in which the proteasome shuttle proteins DNA Damage Inducible 1 (DDI1) and DNA
Damage Inducible 2 (DDI2), function to remove Replication Termination Factor 2 (RTF2) from stalled forks to
mediate a proper recovery. Conversely, depletion of DDI1/2 results in retention of RTF2 in the replisome which
causes a replication fork restart defect that leads to severe genomic instability and cell death. The observed cell
death upon DDI1/2 depletion can be rescued by reducing levels of RTF2 at the replication fork. Overall, our
laboratory has therefore shown that the replisome is reorganized during the replication stress response by the
regulated removal of an essential replisome component.
 In this study, we will elucidate the mechanism by which RTF2 is removed from the replication fork and
determine where RTF2 functions are required in the genome to maintain genome stability. Given that RTF2 is
removed by a proteosome shuttle protein, we hypothesize that the removal of RTF2 requires the ubiquitin
proteasome system. Both ubiquitin conjugation to RTF2 and degradation of the ubiquitin tagged RTF2 must be
tightly regulated and efficient. We have already shown that DDI1/2 regulates the actual removal, but the
mechanism of recognition of RTF2 by DDI1/2 needs to be understood. We propose that DDI1/2 interacts with a
ubiquitin-conjugation on RTF2 to target its degradation and aim to identify (1) the specific residue(s) on RTF2
that must be ubiquitinated for recognition by DDI1/2 and (2) the targeting E3 ligase that ubiquitinates RTF2.
Furthermore, we strive to understand the locations throughout the genome that require the prompt removal of
RTF2 and define the chromatin niche wherein RTF2 functions. Chromatin is an ordered, yet dynamic regulatory
platform composed of DNA and histone proteins and functionally distinct chromatin environments are created,
in part, through reversible covalent post-translational modifications (PTMs) of histones. Our goal is to determine
the genomic locations and chromatin environment that require RTF2 function and identify genomic loci that
require RTF2 removal. With this project, we are studying the fundamental events that coordinate regulation
between the ubiquitin proteosome system (UPS), chromatin, and the replisome at active and stalled replication
forks to promote genome stability.

## Key facts

- **NIH application ID:** 10315162
- **Project number:** 1F32GM143866-01
- **Recipient organization:** ROCKEFELLER UNIVERSITY
- **Principal Investigator:** Penelope Lee Ruiz
- **Activity code:** F32 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $66,390
- **Award type:** 1
- **Project period:** 2021-09-16 → 2023-09-15

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10315162

## Citation

> US National Institutes of Health, RePORTER application 10315162, Understanding the regulatory mechanisms for Replication Termination Factor 2 (RTF2) removal and function during DNA replication (1F32GM143866-01). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10315162. Licensed CC0.

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