The overall goal is to understand the molecular mechanisms that mediate the reactions of DNA replication. Structure studies of the individual proteins, their functional sub-assemblies, and the replisome itself combined with genetic and biochemical analyses will define the protein-protein interactions that coordinate the steps. Studies focus on the replication system derived from phage T7 since its chromosome is replicated in a manner characteristic of those of more complex systems, but by a small number of proteins, most encoded by the phage. The crystal structure of T7 DNA polymerase complexed to a primer-template, a deoxynucleoside triphosphate, and its processivity factor, E. coli thioredoxin, provides insight into polymerization and suggests future studies. The mechanism of processivity conferred by the thioredoxin-thumb subdomain will be pursued by identifying contacts of this subdomain with the DNA and other residues on the polymerise and by examining the movement of the complex on DNA. The gene 4 protein provides helicase and primase activities at the replication fork. The two activities will be studied independently using cloned domains that encode each. The domain responsible for the formation of the functional helicase hexamer will be identified and its interaction with both strands of a duplex DNA substrate will be characterized. Sequence-specific recognition of single-stranded DNA by the primase domain will be studied by determining the role of the CyS4 zinc motif and by identifying the nucleotide binding sites for primer synthesis. The interaction of the helicase and primase with the polymerase will be dissected by use of altered proteins and by suppressor mutation analysis. The T7 gene 2.5 single-strand binding protein interacts with all the T7 replication proteins and is essential for coordination of reactions at a replication fork. The role of the gene 2.5 protein dimer in homologous base-paining and in protein-protein interactions will be examined and its binding to single-stranded DNA will be characterized. The interaction of gene 2.5 protein with DNA polymerase and gene 4 protein will be pursued through the use of in vitro mutagenesis and cross-linking studies. A mini-circle replication system in which leading and lagging strand synthesis are coupled and the lagging strand polymerase operates processively will be used to dissect protein interactions at the fork, particularly those involving gene 2.5 protein. The mechanism by which a replication loop is formed and its relationship to Okazaki fragment size will be pursued. The mini-circle replication system will be used to examine replication phenomena such as the formation of expansion of trinucleotide repeats.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM054397-39
Application #
6490100
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Program Officer
Wolfe, Paul B
Project Start
1976-01-01
Project End
2003-12-31
Budget Start
2002-01-01
Budget End
2002-12-31
Support Year
39
Fiscal Year
2002
Total Cost
$586,209
Indirect Cost
Name
Harvard University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
082359691
City
Boston
State
MA
Country
United States
Zip Code
02115
Hernandez, Alfredo J; Richardson, Charles C (2018) Gp2.5, the multifunctional bacteriophage T7 single-stranded DNA binding protein. Semin Cell Dev Biol :
Hernandez, Alfredo J; Richardson, Charles C (2017) Kinetics of Lagging-strand DNA Synthesis In Vitro by the Bacteriophage T7 Replication Proteins. J Vis Exp :
Hernandez, Alfredo J; Lee, Seung-Joo; Richardson, Charles C (2016) Primer release is the rate-limiting event in lagging-strand synthesis mediated by the T7 replisome. Proc Natl Acad Sci U S A 113:5916-21
Tran, Ngoc Q; Tabor, Stanley; Richardson, Charles C (2014) Genetic requirements for sensitivity of bacteriophage t7 to dideoxythymidine. J Bacteriol 196:2842-50
Mitsunobu, Hitoshi; Zhu, Bin; Lee, Seung-Joo et al. (2014) Flap endonuclease activity of gene 6 exonuclease of bacteriophage T7. J Biol Chem 289:5860-75
Akabayov, Barak; Akabayov, Sabine R; Lee, Seung-Joo et al. (2013) Impact of macromolecular crowding on DNA replication. Nat Commun 4:1615
Zhu, Bin; Tabor, Stanley; Raytcheva, Desislava A et al. (2013) The RNA polymerase of marine cyanophage Syn5. J Biol Chem 288:3545-52
Tran, Ngoc Q; Tabor, Stanley; Amarasiriwardena, Chitra J et al. (2012) Characterization of a nucleotide kinase encoded by bacteriophage T7. J Biol Chem 287:29468-78
Tran, Ngoc Q; Lee, Seung-Joo; Akabayov, Barak et al. (2012) Thioredoxin, the processivity factor, sequesters an exposed cysteine in the thumb domain of bacteriophage T7 DNA polymerase. J Biol Chem 287:39732-41
Zhu, Bin; Lee, Seung-Joo; Tan, Min et al. (2012) Gene 5.5 protein of bacteriophage T7 in complex with Escherichia coli nucleoid protein H-NS and transfer RNA masks transfer RNA priming in T7 DNA replication. Proc Natl Acad Sci U S A 109:8050-5

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