Our goal is to define the molecular mechanisms common to all replication systems. The approach is to characterize the proteins involved in DNA replication using genetic, biochemical, and structural approaches and to identify the functional assemblies that coordinate the reactions. The replication system of bacteriophage T7 is advantageous for this approach since a T7 replisome consists of only a few proteins most of which are encoded by the phage, and yet the replisome functions in a manner similar to those found in more complex systems. Crystal structures of T7 DNA polymerase, T7 helicase-primase, T7 single-stranded DNA (ssDNA) binding protein, and E. coli thioredoxin (processivity factor) provide insight into their functions and suggest future studies. Studies of the contacts of the thioredoxin-binding domain of the thumb of the polymerase with DNA and with residues in the polymerase will elucidate the mechanisms of processivity, DNA partitioning between the polymerase and exonuclease sites, and the role of the thioredoxin binding domain in providing a shared docking site for the replication proteins. The interaction of the gene 2.5 ssDNA binding protein with DNA and with the helicase-primase and polymerase will be examined as will its ability to mediate homologous base pairing. In vitro mutagenesis of the gene 4 helicase will be used to examine oligomerization of the protein, nucleotide binding and hydrolysis, and coupling of nucleotide hydrolysis to unwinding of DNA. The interaction of the Cys4 zinc motif of the gene 4 primase with the catalytic core of the protein to achieve sequence-specific recognition of ssDNA will be studied and the role of the zinc motif in delivering the RNA primer to the polymerase examined. The zinc motif interacts with the RNA polymerase domain of an adjacent subunit within the helicase hexameric assembly. This interaction as well as that of the primase domain with the helicase domain is important in coordinating primase and helicase activities. The four T7 proteins function as a replisome in a mini-circle DNA replication system to mediate coordinated synthesis. Studies using this system will examine the coupling of leading and lagging strand synthesis, the formation of a loop of lagging strand DNA, and the recycling of the lagging strand polymerase.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM054397-44
Application #
7160495
Study Section
Special Emphasis Panel (ZRG1-MBC-2 (01))
Program Officer
Dearolf, Charles R
Project Start
1976-01-01
Project End
2007-12-31
Budget Start
2007-01-01
Budget End
2007-12-31
Support Year
44
Fiscal Year
2007
Total Cost
$631,752
Indirect Cost
Name
Harvard University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
047006379
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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