This is a collaborative project between the Konigsberg and Karam laboratories aimed at understanding the structure and dynamics of DNA replicases. The current focus is the replicative DNA polymerase (gp43) of the T4 family of phages. Phylogenetic, biochemical, structural, and genetic tools are being used to analyze the mechanisms by which the multifunctional gp43 controls 3 categories of function: (a) fidelity of replicative DNA synthesis through accurate base selection and effective editing, (b) translational repression of its own mRNA through a highly specific RNA binding function, and (c) recognition of other replication proteins in the phage replicase. In fidelity studies, the investigators will use complementary biological and biochemical (steady-state and presteady-state kinetic) assay systems to identify gp43 residues that play roles in dNTP discrimination prior to nucleotide transfer. They also plan to identify residues that contribute to the conformational changes that precede chemistry in the nucleotidyl transfer reaction. Their studies draw heavily on knowledge of the crystal structure of RB69 gp43 from phage RB69, a phylogenetic relative of T4. The structure of this enzyme was solved during the previous project period, and they have placed a special emphasis on understanding the structure-function relationships between residues that form a binding pocket for the incoming dNTP and that help to align the complementary base in the template strand so that a sterically constrained space can be formed permitting only the """"""""correctly"""""""" matched dNTP's to fit in the proper geometric orientation for the nucleotidyl transfer reaction. The investigators will attempt to crystallize a RB69 gp43 mutant, Y567A, that is relatively permissive for incorporation of mispaired dNTP's, together with a dideoxy terminated primer-template and mispaired dNTP's. The structure of these complexes should provide insight into the structural requirements for fidelity. The results of the proposed work will contribute to a better understanding of the mechanisms of viral infectious diseases, especially those mediated by viruses that mutate rapidly. It will also provide a structural basis for mutagenic mechanisms that compromise the fidelity of replicative and repair polymerases.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM063276-04
Application #
6722939
Study Section
Biochemistry Study Section (BIO)
Program Officer
Jones, Warren
Project Start
2001-04-01
Project End
2006-03-31
Budget Start
2004-04-01
Budget End
2006-03-31
Support Year
4
Fiscal Year
2004
Total Cost
$305,336
Indirect Cost
Name
Yale University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
043207562
City
New Haven
State
CT
Country
United States
Zip Code
06520
Christian, Thomas V; Konigsberg, William H (2018) Single-molecule FRET reveals proofreading complexes in the large fragment of Bacillus stearothermophilus DNA polymerase I. AIMS Biophys 5:144-154
Vashishtha, Ashwani Kumar; Konigsberg, William H (2018) The effect of different divalent cations on the kinetics and fidelity of Bacillus stearothermophilus DNA polymerase. AIMS Biophys 5:125-143
Vashishtha, Ashwani Kumar; Konigsberg, William H (2016) Effect of Different Divalent Cations on the Kinetics and Fidelity of RB69 DNA Polymerase. Biochemistry 55:2661-70
Xia, Shuangluo; Konigsberg, William H (2014) RB69 DNA polymerase structure, kinetics, and fidelity. Biochemistry 53:2752-67
Xia, Shuangluo; Konigsberg, William H (2014) Mispairs with Watson-Crick base-pair geometry observed in ternary complexes of an RB69 DNA polymerase variant. Protein Sci 23:508-13
Xia, Shuangluo; Wang, Jimin; Konigsberg, William H (2013) DNA mismatch synthesis complexes provide insights into base selectivity of a B family DNA polymerase. J Am Chem Soc 135:193-202
Xia, Shuangluo; Wood, Marcus; Bradley, Michael J et al. (2013) Alteration in the cavity size adjacent to the active site of RB69 DNA polymerase changes its conformational dynamics. Nucleic Acids Res 41:9077-89
Xia, Shuangluo; Vashishtha, Ashwani; Bulkley, David et al. (2012) Contribution of partial charge interactions and base stacking to the efficiency of primer extension at and beyond abasic sites in DNA. Biochemistry 51:4922-31
Englert, Markus; Xia, Shuangluo; Okada, Chiaki et al. (2012) Structural and mechanistic insights into guanylylation of RNA-splicing ligase RtcB joining RNA between 3'-terminal phosphate and 5'-OH. Proc Natl Acad Sci U S A 109:15235-40
Xia, Shuangluo; Christian, Thomas D; Wang, Jimin et al. (2012) Probing minor groove hydrogen bonding interactions between RB69 DNA polymerase and DNA. Biochemistry 51:4343-53

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