The general objective of this proposal are to define the kinetic and structural characteristics of the polymerization process catalyzed by repair and replicative DNA enzymes with emphasis on how replicative fidelity is achieved. Specifically, the minimal reaction sequence for incorporation and misincorporation of deoxynucleotide 5'-triphosphates into template-primers by repair polymerases such as Po1 I and on the other hand to evaluate the influence of the accessory proteins of the T4 replication complex on leading strand synthesis and ultimately at a replication fork. In the case of the HIV reverse transcriptase such a scheme will permit the quantitation of the effects and mode of action of various nucleotide analogs on HIV reverse transcriptase catalyzed replication. The experimental approach is based on rapid quench methods that examine the initial turnover. To compliment studies on the dynamics of these polymerization processes structural studies employing fluorescently labelled DNA, inter strand cross-linked DNA, and photoaffinity labelled DNA will be used to map protein DNA interactions in order to identify specific domains and residues important in both the binding and catalytic processes. The expansion of these methods to the T4 replicative proteins will aid in identification of those proteins that interact with the DNA or with other protein surfaces. The ultimate objective of this work is to elucidate the relationship between polymerase structure and function that results in the enzymes high catalytic efficiency and fidelity.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM013306-27
Application #
3268471
Study Section
Physical Biochemistry Study Section (PB)
Project Start
1976-01-01
Project End
1994-12-31
Budget Start
1992-01-01
Budget End
1992-12-31
Support Year
27
Fiscal Year
1992
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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