# Molecular mechanism of eukaryotic chromosome replication

> **NIH NIH R35** · SLOAN-KETTERING INST CAN RESEARCH · 2024 · $675,277

## Abstract

Summary
 DNA replication stress is a major source for genome instability associated with numerous diseases,
including cancer. Therefore, understanding the molecular basis of replication stress and, conversely, how
accurate, complete, and rapid chromosomal DNA replication is achieved during normal cell proliferation, is
crucial for understanding the mechanisms that maintain or threaten genome stability during normal
development and disease, respectively. Here we propose experiments that will illuminate both the intrinsic
mechanism of the eukaryotic DNA replication machinery and its response to diverse replication stress
conditions. Previously, we have generated a fully reconstituted origin-dependent DNA replication system
based on purified budding yeast proteins. More recently, we have begun to reconstitute replisomes with
purified human proteins. These systems form the central platform for research in our lab.
 The normal and uninterrupted progression of replication forks is frequently challenged by physical
obstacles on the parental DNA. These include non-B-form DNA secondary structures and tightly bound non-
histone protein-DNA complexes. While non-B-form DNA secondary structures induce fork stalling in a
stochastic and unscheduled manner, the programmed stalling of the replisome at tightly bound protein-DNA
complexes can serve important biological functions. How individual physical obstacles on chromosomal DNA
induce fork stalling, are overcome by fork restoration mechanisms, or elicit distinct biological outcomes is
incompletely understood. To illuminate fork stalling mechanisms, we will investigate the molecular
mechanisms by which such physical obstacles impede the eukaryotic replicative DNA helicase, CMG (Cdc45-
MCM-GINS). Specifically, we will determine the mechanisms by which G-quadruplexes (G4s) impede CMG
progression on the leading strand and the mechanisms by which replication forks stalled at G4s may be
restored by accessory DNA helicases and fork remodelers. As an example for programmed fork stalling at
protein-DNA complexes, we will investigate the mechanism of unidirectional fork stalling at the replication fork
barrier (RFB) derived from both yeast and human rDNA repeats and examine the mechanism of replication
termination upon fork convergence at these sites. Stalled replication forks are both inducers and targets of
the replication checkpoint, which is essential for the maintenance of genome integrity by stabilizing stalled
forks and coordinating fork restoration with cell cycle progression. Continuing our previous work, which has
led to the identification of yeast replisomes as direct targets for the checkpoint effector kinase, Rad53, we will
determine the mechanism(s) by which the checkpoint controls fork progression.

## Key facts

- **NIH application ID:** 10765321
- **Project number:** 1R35GM152094-01
- **Recipient organization:** SLOAN-KETTERING INST CAN RESEARCH
- **Principal Investigator:** Dirk Remus
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $675,277
- **Award type:** 1
- **Project period:** 2024-08-10 → 2029-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10765321

## Citation

> US National Institutes of Health, RePORTER application 10765321, Molecular mechanism of eukaryotic chromosome replication (1R35GM152094-01). Retrieved via AI Analytics 2026-06-12 from https://api.ai-analytics.org/grant/nih/10765321. Licensed CC0.

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