The thrust of this proposal is to elaborate the mechanism of action of various enzymes that catalyze differing phosphoryl transfer reactions. The experimental approach projects an interplay between complimentary nonenzymic and enzymic investigations with the former serving to define features of possible transition states and the latter designed to elaborate the steps and associated energetics of the reaction sequence, the existence and structure of enzyme bound intermediates, and the nature as well as influence on key steps of enzyme-substrate interactions. The acquired insights should broaden our general perspective of enzymic catalysis. The nonenzymic studies focus on the reactions of thiophosphate monoesters; the enzymic investigations encompass the following activities: the polymerization of nucleoside diphosphates catalyzed by primer independent polynucleotide phosphorylase; the 3'-elongation of a DNA template-primer as well as degradation of the duplex via 3'-5' hydrolysis or pyrophosphorolysis promoted by the multiple activities of DNA polymerase I; and the site specific hydrolytic cleavage accomplished by the endonuclease, EcoRI. Since crystallographic studies now have revealed structural features of the latter two enzymes, experiments have been emphasized that correlate catalytic function with structure. Specific approaches to be employed include pre-steady state and isotope-partitioning kinetic methods, variations in both substrate and protein structures, and distance measurements within substrate enzyme complexes. It is hoped that the results of the proposed studies are also generally applicable to understanding nonchemical phenomenona such as processivity, translocation, and the mode of sequence searching employed by these enzymes.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM013306-22
Application #
3268467
Study Section
Biochemistry Study Section (BIO)
Project Start
1976-01-01
Project End
1989-12-31
Budget Start
1987-01-01
Budget End
1987-12-31
Support Year
22
Fiscal Year
1987
Total Cost
Indirect Cost
Name
Pennsylvania State University
Department
Type
Schools of Arts and Sciences
DUNS #
City
University Park
State
PA
Country
United States
Zip Code
16802
Benkovic, Stephen J; Spiering, Michelle M (2017) Understanding DNA replication by the bacteriophage T4 replisome. J Biol Chem 292:18434-18442
Hedglin, Mark; Aitha, Mahesh; Benkovic, Stephen J (2017) Monitoring the Retention of Human Proliferating Cell Nuclear Antigen at Primer/Template Junctions by Proteins That Bind Single-Stranded DNA. Biochemistry 56:3415-3421
Hedglin, Mark; Benkovic, Stephen J (2017) Eukaryotic Translesion DNA Synthesis on the Leading and Lagging Strands: Unique Detours around the Same Obstacle. Chem Rev 117:7857-7877
Hedglin, Mark; Benkovic, Stephen J (2017) Replication Protein A Prohibits Diffusion of the PCNA Sliding Clamp along Single-Stranded DNA. Biochemistry 56:1824-1835
Spiering, Michelle M; Hanoian, Philip; Gannavaram, Swathi et al. (2017) RNA primer-primase complexes serve as the signal for polymerase recycling and Okazaki fragment initiation in T4 phage DNA replication. Proc Natl Acad Sci U S A 114:5635-5640
Hedglin, Mark; Pandey, Binod; Benkovic, Stephen J (2016) Characterization of human translesion DNA synthesis across a UV-induced DNA lesion. Elife 5:
Hedglin, Mark; Pandey, Binod; Benkovic, Stephen J (2016) Stability of the human polymerase ? holoenzyme and its implications in lagging strand DNA synthesis. Proc Natl Acad Sci U S A 113:E1777-86
Choi, Jung-Suk; Dasari, Anvesh; Hu, Peter et al. (2016) The use of modified and non-natural nucleotides provide unique insights into pro-mutagenic replication catalyzed by polymerase eta. Nucleic Acids Res 44:1022-35
Noble, Erin; Spiering, Michelle M; Benkovic, Stephen J (2015) Coordinated DNA Replication by the Bacteriophage T4 Replisome. Viruses 7:3186-200
Zhao, Yanhui; Chen, Danqi; Yue, Hongjun et al. (2014) Dark-field illumination on zero-mode waveguide/microfluidic hybrid chip reveals T4 replisomal protein interactions. Nano Lett 14:1952-60

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