Understanding the enzymatic mechanisms of DNA replication is an important problem at the foundation of molecular biology. The overarching goal of our studies is to develop a quantitative model of DNA replication that will accurately recapitulate the catalytic properties of the enzymes (helicase, polymerase, primase) and the functional coupling between them during the reactions of DNA unwinding, synthesis, and priming. Our work will advance the field of DNA replication by providing detailed insights into how helicase and polymerase work together and how leading and lagging strand synthesis are coupled. We propose studies of replicative enzymes of the bacteriophage T7 and the human mitochondria. Phage T7 encodes one of the simplest replication systems that provide a model for human mitochondrial DNA replication as well as more complicated systems. Defects in helicase and polymerase functions are associated with diseases such as cancer, premature ageing, and neuromuscular disorders. A detailed understanding of the enzymatic mechanisms of helicase and polymerase mechanisms will enable development of therapeutics for such diseases. Quantitative transient state and single molecule kinetics studies allowed us to discover new mechanisms and synergies between the T7 replicative enzymes. A major goal with the T7 studies is to understand the collaborative coupling between the replicative enzymes during leading and lagging strand DNA synthesis. A major goal of the mitochondrial studies is to reconstitute replication in vitro and characterize select disease- causing mutants of Twinkle. This proposal builds upon our characterization of T7 and mitochondria replication from the last grant cycle to address key questions that arose from prior results. How helicase and polymerase are physically and functionally coupled at the replication fork? What is the base pair stepping mechanism of the helicase-polymerase? How does collaborative coupling affect DNA synthesis fidelity (misincorporation, proofreading)? What is the mechanism of lagging strand DNA synthesis? How do specific disease-related point mutations in Twinkle change its function compared to wild-type? What is the role of the strand exchange activity of Twinkle? These fundamental questions on DNA replication will be addressed in the following specific aims: 1) Investigate coupling between the activities of helicase and polymerase during leading strand DNA synthesis. 2) Investigate mechanisms of lagging strand DNA synthesis. 3) Investigate mechanisms of wild-type and mutant helicase Twinkle and polymerase.

Public Health Relevance

This work will be invaluable for the development of helicase/polymerase targeted therapy for cancer and mitochondrial related disorders. More generally, this project will advance the field of DNA replication by providing important insights into how replicative enzymes work and how their activities are coupled to faithfully copy genomes in a timely manner. The studies will provide basic knowledge that is necessary to understand the diseases at the molecular level and strategies for prevention and treatment.

Agency
National Institute of Health (NIH)
Type
Research Project (R01)
Project #
5R01GM055310-20
Application #
8739656
Study Section
Macromolecular Structure and Function E Study Section (MSFE)
Program Officer
Barski, Oleg
Project Start
Project End
Budget Start
Budget End
Support Year
20
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Rbhs-Robert Wood Johnson Medical School
Department
Biochemistry
Type
Schools of Medicine
DUNS #
City
Piscataway
State
NJ
Country
United States
Zip Code
08854
Syed, Salman; Pandey, Manjula; Patel, Smita S et al. (2014) Single-molecule fluorescence reveals the unwinding stepping mechanism of replicative helicase. Cell Rep 6:1037-45
Pandey, Manjula; Patel, Smita S (2014) Helicase and polymerase move together close to the fork junction and copy DNA in one-nucleotide steps. Cell Rep 6:1129-38
Sun, Bo; Johnson, Daniel S; Patel, Gayatri et al. (2011) ATP-induced helicase slippage reveals highly coordinated subunits. Nature 478:132-5
Patel, Smita S; Pandey, Manjula; Nandakumar, Divya (2011) Dynamic coupling between the motors of DNA replication: hexameric helicase, DNA polymerase, and primase. Curr Opin Chem Biol 15:595-605
Levin, Mikhail K; Hingorani, Manju M; Holmes, Raquell M et al. (2009) Model-based global analysis of heterogeneous experimental data using gfit. Methods Mol Biol 500:335-59
Pandey, Manjula; Syed, Salman; Donmez, Ilker et al. (2009) Coordinating DNA replication by means of priming loop and differential synthesis rate. Nature 462:940-3
Rajagopal, Vaishnavi; Patel, Smita S (2008) Single strand binding proteins increase the processivity of DNA unwinding by the hepatitis C virus helicase. J Mol Biol 376:69-79
Stano, Natalie M; Jeong, Yong-Joo; Donmez, Ilker et al. (2005) DNA synthesis provides the driving force to accelerate DNA unwinding by a helicase. Nature 435:370-3