# Biochemical Mechanism and Structure of the Eukaryotic Replication Fork

> **NIH NIH R01** · ROCKEFELLER UNIVERSITY · 2021 · $339,000

## Abstract

PROJECT SUMMARY
Duplication of the genome is essential to all cells. Yet very few labs have succeeded in reconstituting the
enzymology of the eukaryotic replication fork from pure proteins for mechanistic studies. This is in part due to
the numerous proteins required to drive replication and the difficulty in obtaining these many factors. Unlike
bacteria, eukaryotes use different DNA polymerases to duplicate the leading and lagging strands, the
eukaryotic helicase contains 11 distinct proteins, and there exist numerous eukaryotic replication proteins that
have no homologue in bacteria.
We have made several breakthrough studies using pure proteins to reconstitute the eukaryotic budding yeast
(Saccharomyces cerevisiae) replisome (>30 different subunits). We have determined the organization of
replisome proteins by EM, the mechanisms by which DNA polymerase (Pol) ε is directed to the leading strand
and Pol δ is directed to the lagging strand, along with quality control mechanisms that prevent these Pols from
working on the “wrong” strands. We also solved the orientation of the CMG helicase while translocating on
DNA by cryoEM and the consequent profound implications for origin initiation. Questions to be addressed in
this proposal include development of the Homo sapiens (H.s.) replisome machinery to address how metazoan
replisomes function to perform fork regression, a genome stability process in higher eukaryotes. Thus far we
have purified the difficult multisubunit recombinant H.s. factors, including 11-subunit H.s. CMG helicase and
have reconstituted the H.s. leading strand replisome. We will characterize how the H.s. replisome functions,
compare it to budding yeast and then examine metazoan specific ATPase fork remodelers that reverse stalled
forks for genomic integrity. Fork remodelers have not been studied with replisome proteins, and we will
address their action in the presence of H.s. replisome proteins and also the fork protection factors, H.s. RAD51
recombinase and the BRCA2 tumor suppressor. We will also examine how reversed forks with bound BRCA2-
RAD51, are restored to enable replication restart. We also propose to continue our understanding of replisome
structure through cryoEM of various replisome subcomplexes in both the human and budding yeast systems.
In overview, the studies proposed here will provide a deep understanding of the workings of the eukaryotic
DNA replication machinery, and its intimate involvement in DNA repair, mutagenesis, and human disease.
Replication is also crucial for a positive response to many anticancer drugs that are currently in use. Hence
detailed knowledge of this central and vital process to cellular life will provide important information useful to
prevention and cure of human disease.

## Key facts

- **NIH application ID:** 10150468
- **Project number:** 5R01GM115809-07
- **Recipient organization:** ROCKEFELLER UNIVERSITY
- **Principal Investigator:** MICHAEL E O'DONNELL
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $339,000
- **Award type:** 5
- **Project period:** 2015-08-01 → 2023-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10150468

## Citation

> US National Institutes of Health, RePORTER application 10150468, Biochemical Mechanism and Structure of the Eukaryotic Replication Fork (5R01GM115809-07). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10150468. Licensed CC0.

---

*[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*