The herpes simplex virus type 1 DNA polymerase processivity factor increases fidelity without altering pre-steady-state rate constants for polymerization or excision.
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 nm for Pol and 7.0 nm 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 ( approximately 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.
Chaudhuri
Murari
M
Department of Molecular Virology, Immunology, and Medical Genetics, Ohio State University, Columbus, Ohio 43210, USA.
Song
Liping
L
Parris
Deborah S
DS
D000818:Animals; D020137:Base Pair Mismatch; D001483:Base Sequence; D001665:Binding Sites; D020134:Catalytic Domain; D002460:Cell Line; D004247:DNA / Q000096:biosynthesis / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000502:physiology*; D004305:Dose-Response Relationship, Drug; D005090:Exodeoxyribonucleases / Q000737:chemistry* / Q000502:physiology*; D007313:Insecta; D007700:Kinetics; D008969:Molecular Sequence Data; D013696:Temperature; D013942:Thymine Nucleotides / Q000494:pharmacology; D013997:Time Factors; D014764:Viral Proteins / Q000737:chemistry* / Q000502:physiology*
D013942:Thymine Nucleotides; D014764:Viral Proteins; D004247:DNA; D004259:DNA-Directed DNA Polymerase; D005090:Exodeoxyribonucleases; C043417:DNA polymerase, Simplexvirus; C024157:thymidine 5'-triphosphate