We are investigating the structures and catalytic mechanisms of the enzymes from bacteriophage T7 that catalyze DNA replication. The experiments described in this application will focus on the interactions of these replication proteins with one another and with DNA in the context of a replication fork. The T7 replisome is a model system for understanding the dynamic commerce of DNA replication-the unwinding of two DNA strands, the continuous synthesis of DNA on the leading strand of the replication fork, the priming of Okazaki fragments and discontinuous synthesis of DNA on the lagging strand. These features are universal for all DNA-based life forms, and they are most easily examined at the molecular level in a simple model replication system like that encoded by phage T7. In spite of the cartoons of replication forks that are shown in college textbooks, very little is actually known about the overall organization and molecular architecture of any replication system. Our goal is to identify and characterize the transient and stable protein-protein and protein-DNA interactions that underlie the coupled synthesis of two antiparallel DNA strands. These processes are fundamental for the growth and development of humans and other organisms. Unique features of the replication process in prokaryotes are attractive targets for the development of selective antimicrobial agents to treat infectious diseases. ? ? ?

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Special Emphasis Panel (ZRG1-BPC-B (02))
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Lewis, Catherine D
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Washington University
Schools of Medicine
Saint Louis
United States
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Wallen, Jamie R; Zhang, Hao; Weis, Caroline et al. (2017) Hybrid Methods Reveal Multiple Flexibly Linked DNA Polymerases within the Bacteriophage T7 Replisome. Structure 25:157-166
Wallen, Jamie R; Majka, Jerzy; Ellenberger, Tom (2013) Discrete interactions between bacteriophage T7 primase-helicase and DNA polymerase drive the formation of a priming complex containing two copies of DNA polymerase. Biochemistry 52:4026-36
Raschle, Markus; Knipscheer, Puck; Knipsheer, Puck et al. (2008) Mechanism of replication-coupled DNA interstrand crosslink repair. Cell 134:969-80
Kim, Tae Woo; Brieba, Luis G; Ellenberger, Tom et al. (2006) Functional evidence for a small and rigid active site in a high fidelity DNA polymerase: probing T7 DNA polymerase with variably sized base pairs. J Biol Chem 281:2289-95
Brieba, Luis G; Kokoska, Robert J; Bebenek, Katarzyna et al. (2005) A lysine residue in the fingers subdomain of T7 DNA polymerase modulates the miscoding potential of 8-oxo-7,8-dihydroguanosine. Structure (Camb) 13:1653-9
Dutta, Shuchismita; Li, Ying; Johnson, Donald et al. (2004) Crystal structures of 2-acetylaminofluorene and 2-aminofluorene in complex with T7 DNA polymerase reveal mechanisms of mutagenesis. Proc Natl Acad Sci U S A 101:16186-91
Brieba, Luis G; Eichman, Brandt F; Kokoska, Robert J et al. (2004) Structural basis for the dual coding potential of 8-oxoguanosine by a high-fidelity DNA polymerase. EMBO J 23:3452-61
Kato, Masato; Ito, Takuhiro; Wagner, Gerhard et al. (2004) A molecular handoff between bacteriophage T7 DNA primase and T7 DNA polymerase initiates DNA synthesis. J Biol Chem 279:30554-62
Li, Ying; Dutta, Shuchismita; Doublie, Sylvie et al. (2004) Nucleotide insertion opposite a cis-syn thymine dimer by a replicative DNA polymerase from bacteriophage T7. Nat Struct Mol Biol 11:784-90
van Oijen, Antoine M; Blainey, Paul C; Crampton, Donald J et al. (2003) Single-molecule kinetics of lambda exonuclease reveal base dependence and dynamic disorder. Science 301:1235-8

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