Investigation of the electrostatic interactions of the T7 replisome

WCU Author/Contributor (non-WCU co-authors, if there are any, appear on document)
Brittni M. Foster (Creator)
Western Carolina University (WCU )
Web Site:
Jamie R. Wallen

Abstract: DNA replication is a process in which genetic information is copied and passed on from generationto generation. Multiple proteins of specialized function must transiently assembleand communicate to rapidly and accurately copy DNA. We study replication in bacteriophageT7, a virus that infects Escherichia coli (E. coli ), because it is a relatively simplemodel system that recapitulates all essential activities seen in more complex systems in justfour proteins. Recently our laboratory determined a crystal structure of an electrostaticinteraction between T7 primase-helicase and DNA polymerase, and the structure revealedthe acidic C-terminal tail of the T7 primase-helicase binds to a basic patch on the surface ofthe polymerase. The C-terminal tail of the primase-helicase contains a phenylalanine residuethat is essential for binding polymerase, and our structure reveals that this phenylalaninebinds to a hydrophobic cleft near the basic patch. Our crystal structure also unexpectedlyrevealed an interaction between a nucleotide triphosphate and tryptophan 160 located in theexonuclease active site, and this interaction has shed light on how the exonuclease domainof T7 polymerase recognizes a DNA substrate. To validate our crystal structure, point mutationswere generated along the basic patch of the polymerase as well as the hydrophobicpocket to disrupt the observed interactions.Using in vivo methods such as phage complementation and multi-step growth curvewe are able to directly test the effects on the virus replication during infection of E. coli. Phage complementation results demonstrate that these mutations F487A, I569A, R687A andR590 A show a 3-log and 4-log difference in growth compared to WT. Plaque assay resultsshow that R590 A is critical for replication, as the virus shows no growth under identical conditionsfor WT. The other mutations I569A, I569A, and F487A were also impaired duringthe plaque assay and similar to R590 A showed no plaque formation. R687A showed substantiallysmaller plaques compared to WT and we were unable to calculate a titer. No plaqueformation and smaller plaques indicate that the virus is heavily impaired in DNA replication.Under identical conditions to WT, R590 A show no growth over a 15 hour period where asWT reaches a maximum amount of virus and plateaus at 4 hours. Preliminary data forI569A and F487A show these mutations also result in impaired and reach a lower maximumviral concentration compared to WT. In the crystal structure R590 of the polymerase makesa critical contact with the C-terminal tail, and in agreement with the structure, mutation of R590 to alanine suggests that this residue serves an essential function in DNA replication. Phage complementation results for the two mutations in the exonuclease active site,W160 a and 5A7A, gave similar results compared to WT at a log of 1x1012 PFU/mL. Thedouble mutation D5A and E7A (5A7A) is unable to coordinate Mg+2 causing exonucleaseactivity to be diminished. Results for rolling circle reveal that polymerase with a 5A7Amutation can perform strand displacement synthesis without helicase. Future work willfocus on continuing Rolling circle, growth curve, and primer extension assays.

Additional Information

Language: English
Date: 2016
antibiotic resistance, Phage, Protein, T7
Bacteriophages -- Genetics
DNA replication
Viral genetics
Microbial genetics

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