Duplication of the DNA genome is essential to the life of all cells. To date, no other lab has succeeded in reconstituting the enzymology of the eukaryotic replication fork. This is in part due to the numerous proteins required and the difficulty in obtaining them. Unlike bacteria, eukaryotes use different multiprotein DNA polymerases to duplicate the two (leading and lagging) strands of DNA. The eukaryotic helicase is composed of 11 distinct proteins, while bacteria simply use a homohexamer. In addition, there are numerous proteins that function at eukaryotic replication forks that have no homologue in bacteria. We have recently succeeded in reconstituting a eukaryotic (Saccharomyces cerevisiae) replisome from pure proteins. The reconstituted replisome utilizes 31 distinct proteins to duplicate both leading and lagging strands of one DNA molecule. The reconstituted process requires the 11-subunit helicase among several other factors, and recapitulates the asymmetric utilization of the distinctive polymerases in eukaryotic cells. Thus only DNA polymerase epsilon (Pol e) functions on the leading strand and DNA polymerase delta (Pol d) functions on the lagging strand. These Pols are prevented from working on the wrong strand in the reconstituted system. Questions to be addressed in this proposal include: What mechanism prevents Pol d function on the leading strand, and what prevents Pol e from working on the lagging strand? The reconstituted fork travels 5-10 fold slower than forks inside cells, but we are missing many proteins that are known to travel with forks. We will purify these other factors and examine their function in moving replisomes. Do one or more of them speed the fork? Do they affect lagging strand priming frequency, replisome processivity, or replisome stability? We find that many replisome proteins associate to form a large assembly, even in the absence of DNA. The smallest of these is a 15 subunit helicase-Pol e complex. We have solved a 3D EM structure of this leading strand replisome complex in collaboration with Dr. Huilin Li (Brookhaven National Labs), and it shows several unexpected features. We have reconstituted larger complexes as well. We propose to solve the 3D structures of these complexes using cryoEM, as well as collaborate with Dr. Brian Chait (Rockefeller University) to identify protein connectivity t high resolution within the complexes by new innovative cross-linking/mass spectrometry techniques. In overview, the studies proposed here will provide a deep understanding of the workings of the eukaryotic DNA replication machinery. This process is intimately involved 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 that will be useful t prevention and cure of human disease, and maintenance of a healthy state.

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 a eukaryotic cell from pure proteins for the first time, and this project will study the detaile process of how it works, thus gaining intelligence that directly applies to human health and disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM115809-01
Application #
8943081
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Reddy, Michael K
Project Start
2015-08-01
Project End
2019-04-30
Budget Start
2015-08-01
Budget End
2016-04-30
Support Year
1
Fiscal Year
2015
Total Cost
Indirect Cost
Name
Rockefeller University
Department
Biochemistry
Type
Graduate Schools
DUNS #
071037113
City
New York
State
NY
Country
United States
Zip Code
10065
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

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