# Investigating sliding clamps and their contribution to genome stability

> **NIH NIH F31** · UNIV OF MASSACHUSETTS MED SCH WORCESTER · 2022 · $31,746

## Abstract

Project Summary
 All cells must replicate their genome once per cell cycle. To ensure proper duplication, cells integrate
hundreds of factors that copy, surveil, and repair our genetic information. Proliferating Cell Nuclear Antigen
[PCNA] and Rad9-Rad1-Hus1 [9-1-1] are ring-shaped clamps that act as master “conductors” that regulate many
of the factors that replicate and maintain our DNA. PCNA is a homotrimeric ring that coordinates the replisome
during DNA synthesis to work in tandem with DNA repair, chromatin remodeling, and cell cycle progression.
When cells experience dsDNA breaks, they use the heterotrimeric clamp 9-1-1 to coordinate specific “SOS”
repair factors. The collaborative efforts of both clamps are critical for genome stability. Many cancers are linked
to inappropriate clamp coordination and changes in their expression. Because sliding clamps are central to many
oncogenic pathways, we must address how they regulate themselves and their client partners. This proposal
aims to address the following questions about sliding clamps: 1) How do sliding clamps coordinate their various
partners? 2) Does the time sliding clamps spend on DNA influence genome stability? and 3) What determines
site-specific loading of sliding clamps? I propose a multidisciplinary approach to address these questions about
sliding clamps by investigating two-disease causing PCNA variants [PCNA-S228I [serine to isoleucine] and
PCNA-C148S [cysteine to serine]] and the loading mechanism of 9-1-1. I hypothesize that sliding clamps
control genome integrity via site-specific loading, proper partner interactions, and residence-time on
DNA. I further hypothesize that PCNA-S228I and PCNA-C148S disrupt genome integrity by either
promoting premature DNA dissociation or disrupting partner interactions. Finally, I hypothesize that the
Rad17 subunit alters the clamp loader structure to specifically load the 9-1-1 clamp at sites of DNA
damage. In aims 1 and 2, I will use PCNA-S228I and PCNA-C148S to address how clamps “choose” their
partners and regulate their time on DNA. I will use x-ray crystallography, unfolding experiments, and a series of
functional assays to determine how each variant compromises genome stability. In aim 3, I will determine the
loading mechanism of clamp 9-1-1 to address how clamps are loaded to specific sites in the genome. I will use
cryo-electron microscopy to determine how Rad17-RFC binds to clamp 9-1-1. Collectively, my work will broaden
our insight into the factors that cause genome instability which may augment the development of personalized
chemotherapeutics.

## Key facts

- **NIH application ID:** 10373968
- **Project number:** 5F31CA254328-02
- **Recipient organization:** UNIV OF MASSACHUSETTS MED SCH WORCESTER
- **Principal Investigator:** Joseph Magrino
- **Activity code:** F31 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $31,746
- **Award type:** 5
- **Project period:** 2021-03-14 → 2024-03-13

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10373968

## Citation

> US National Institutes of Health, RePORTER application 10373968, Investigating sliding clamps and their contribution to genome stability (5F31CA254328-02). Retrieved via AI Analytics 2026-05-28 from https://api.ai-analytics.org/grant/nih/10373968. Licensed CC0.

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