The Herpes Simplex Virus Type 1 DNA Polymerase Processivity Factor Increases Fidelity without Altering Pre-steady-state Rate Constants for Polymerization or Excision

• Published in: The Journal of biological chemistry 2003-03, Vol.278 (11), p.8996-9004
• Main Authors: Chaudhuri, Murari, Song, Liping, Parris, Deborah S
• Format: Article
• Language: English
• Subjects: Animals; Base Pair Mismatch; Base Sequence; Binding Sites; Catalytic Domain; Cell Line; DNA - biosynthesis; DNA - metabolism; DNA-Directed DNA Polymerase - chemistry; DNA-Directed DNA Polymerase - physiology; Dose-Response Relationship, Drug; Exodeoxyribonucleases - chemistry; Exodeoxyribonucleases - physiology; Insecta; Kinetics; Molecular Sequence Data; Temperature; Thymine Nucleotides - pharmacology; Time Factors; Viral Proteins - chemistry; Viral Proteins - physiology
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M210023200

Pre-steady-state and steady-state kinetics of nucleotide incorporation and excision were used to assess potential mechanisms by which the fidelity of the herpes simplex virus type 1 DNA polymerase catalytic subunit (Pol) is enhanced by its processivity factor, UL42. UL42 had no effect on the pre-steady-state rate constant for correct nucleotide incorporation (150 s −1 ) nor on the primary rate-limiting conformational step. However, the equilibrium dissociation constant for the enzyme in a stable complex with primer-template was 44 n m for Pol and 7.0 n m for Pol/UL42. The catalytic subunit and holoenzyme both selected against incorrect nucleotide incorporation predominantly at the level of nucleotide affinity, although UL42 slowed by 4-fold the maximum rate of incorporation of incorrect, compared with correct, nucleotide. Pol, with or without UL42, cleaved matched termini at a slower rate than mismatched ones, but UL42 did not significantly alter the pre-steady-state rate constant for mismatch excision (∼16 s −1 ). The steady-state rate constant for nucleotide addition was 0.09 s −1 and 0.03 s −1 for Pol and Pol/UL42, respectively, and enzyme dissociation was the rate-limiting step. The longer half-life for DNA complexes with Pol/UL42 (23 s) compared with that with Pol (8 s) affords a greater probability for excision when a misincorporation event does occur, accounting predominantly for the failure of Pol/UL42 to accumulate mismatched product at moderate nucleotide concentrations.