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DNA binding properties and processive proofreading of herpes simplex virus type 1 DNA polymerase
1Forschungsschwerpunkt Genomforschung und Bioinformatik H0601, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 506, D-69120 Heidelberg, Germany.
Biochimica Et Biophysica Acta
|December 22, 1998
Summary
Herpes simplex virus type 1 DNA polymerase (HSV pol) binds DNA with protruding 5' termini and switches between exonuclease and polymerase activity during a single binding event, supporting its role as a replicase.
Area of Science:
- Molecular Biology
- Virology
- Enzymology
Background:
- Herpes simplex virus type 1 DNA polymerase (HSV pol) is an alpha-like DNA polymerase crucial for viral replication.
- Understanding its DNA binding and enzymatic properties is key to elucidating viral DNA synthesis mechanisms.
Purpose of the Study:
- To investigate the DNA binding characteristics of HSV pol.
- To characterize the relationship between DNA binding and the enzymatic activities (polymerase and exonuclease) of HSV pol.
Main Methods:
- Optimized band-shift assay to study DNA binding.
- Polymerase activity assays using poly(dT) to determine optimal primer length.
- Polymerase trap method and high-resolution polyacrylamide gel electrophoresis to analyze enzymatic mode switching.
Main Results:
- HSV pol preferentially binds to double-stranded DNA (dsDNA) with protruding 5'-phosphoryl termini.
- Optimal primer length for HSV pol polymerization activity is 8-10 nucleotides.
- HSV pol can switch between 3',5' exonuclease and polymerase activity within a single DNA binding event, without dissociating from the template.
Conclusions:
- HSV pol exhibits specific DNA binding preferences.
- The ability of HSV pol to proofread via its exonuclease activity while remaining bound supports its function as a processive viral replicase.