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)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM055310-19
Application #
8506129
Study Section
Macromolecular Structure and Function E Study Section (MSFE)
Program Officer
Barski, Oleg
Project Start
1997-01-01
Project End
2017-07-31
Budget Start
2013-09-23
Budget End
2014-07-31
Support Year
19
Fiscal Year
2013
Total Cost
$341,321
Indirect Cost
$109,380
Name
Rbhs-Robert Wood Johnson Medical School
Department
Biochemistry
Type
Schools of Medicine
DUNS #
078795875
City
Piscataway
State
NJ
Country
United States
Zip Code
08854
Sen, Doyel; Patel, Gayatri; Patel, Smita S (2016) Homologous DNA strand exchange activity of the human mitochondrial DNA helicase TWINKLE. Nucleic Acids Res 44:4200-10
Brennan, Lucy D; Forties, Robert A; Patel, Smita S et al. (2016) DNA looping mediates nucleosome transfer. Nat Commun 7:13337
Nandakumar, Divya; Patel, Smita S (2016) Methods to study the coupling between replicative helicase and leading-strand DNA polymerase at the replication fork. Methods 108:65-78
Chang, Han-Wen; Pandey, Manjula; Kulaeva, Olga I et al. (2016) Overcoming a nucleosomal barrier to replication. Sci Adv 2:e1601865
Pandey, Manjula; Elshenawy, Mohamed M; Jergic, Slobodan et al. (2015) Two mechanisms coordinate replication termination by the Escherichia coli Tus-Ter complex. Nucleic Acids Res 43:5924-35
Nandakumar, Divya; Patel, Smita S (2015) Finding the right match fast. Cell 160:809-11
Nandakumar, Divya; Pandey, Manjula; Patel, Smita S (2015) Cooperative base pair melting by helicase and polymerase positioned one nucleotide from each other. Elife 4:
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
Sen, Doyel; Nandakumar, Divya; Tang, Guo-Qing et al. (2012) Human mitochondrial DNA helicase TWINKLE is both an unwinding and annealing helicase. J Biol Chem 287:14545-56

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