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) ? is directed to the leading strand and Pol ? 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.

Public Health Relevance

DNA replication is a central point at which the cell cycle is regulated, and gone awry can lead to cancer. Indeed, several types of cancers have mutations in genes that act with replication fork proteins, and many cancer therapies in common use today require replication for the drugs to take effect. We have reconstituted the replisome machinery of human and budding yeast from pure proteins, and this project will study the detailed process of fork progression and also fork regression protection by the human BRCA2 tumor suppressor, thus gaining intelligence applicable to human health and disease.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
Project #
Application #
Study Section
Molecular Genetics B Study Section (MGB)
Program Officer
Reddy, Michael K
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Rockefeller University
Graduate Schools
New York
United States
Zip Code
Yao, Nina Y; O'Donnell, Mike E (2018) Replication fork convergence at termination: A multistep process. Proc Natl Acad Sci U S A 115:237-239
O'Donnell, Michael E; Li, Huilin (2018) The ring-shaped hexameric helicases that function at DNA replication forks. Nat Struct Mol Biol 25:122-130
Li, Huilin; O'Donnell, Michael E (2018) The Eukaryotic CMG Helicase at the Replication Fork: Emerging Architecture Reveals an Unexpected Mechanism. Bioessays 40:
Bai, Lin; Yuan, Zuanning; Sun, Jingchuan et al. (2017) Architecture of the Saccharomyces cerevisiae Replisome. Adv Exp Med Biol 1042:207-228
Schroeder, Jeremy W; Randall, Justin R; Hirst, William G et al. (2017) Mutagenic cost of ribonucleotides in bacterial DNA. Proc Natl Acad Sci U S A 114:11733-11738
Lewis, Jacob S; Spenkelink, Lisanne M; Schauer, Grant D et al. (2017) Single-molecule visualization of Saccharomyces cerevisiae leading-strand synthesis reveals dynamic interaction between MTC and the replisome. Proc Natl Acad Sci U S A 114:10630-10635
Schauer, Grant; Finkelstein, Jeff; O'Donnell, Mike (2017) In vitro Assays for Eukaryotic Leading/Lagging Strand DNA Replication. Bio Protoc 7:
Yurieva, Olga; Nikiforov Jr, Vadim; Nikiforov, Vadim et al. (2017) Insights into RNA polymerase catalysis and adaptive evolution gained from mutational analysis of a locus conferring rifampicin resistance. Nucleic Acids Res 45:11327-11340
Schauer, Grant D; O'Donnell, Michael E (2017) Quality control mechanisms exclude incorrect polymerases from the eukaryotic replication fork. Proc Natl Acad Sci U S A 114:675-680
Yao, Nina Y; O'Donnell, Mike (2017) DNA Replication: How Does a Sliding Clamp Slide? Curr Biol 27:R174-R176

Showing the most recent 10 out of 24 publications