title: Pre-steady-state Kinetic Analysis of a Family D DNA Polymerase from Thermococcus sp. 9°N Reveals Mechanisms for Archaeal Genomic Replication and Maintenance.
abstract: Family D DNA polymerases (polDs) have been implicated as the major replicative polymerase in archaea, excluding the Crenarchaeota branch, and bear little sequence homology to other DNA polymerase families. Here we report a detailed kinetic analysis of nucleotide incorporation and exonuclease activity for a Family D DNA polymerase from Thermococcus sp. 9°N. Pre-steady-state single-turnover nucleotide incorporation assays were performed to obtain the kinetic parameters, kpol and Kd, for correct nucleotide incorporation, incorrect nucleotide incorporation, and ribonucleotide incorporation by exonuclease-deficient polD. Correct nucleotide incorporation kinetics revealed a relatively slow maximal rate of polymerization (kpol ∼ 2.5 s(-1)) and especially tight nucleotide binding (Kd (dNTP) ∼ 1.7 μm), compared with DNA polymerases from Families A, B, C, X, and Y. Furthermore, pre-steady-state nucleotide incorporation assays revealed that polD prevents the incorporation of incorrect nucleotides and ribonucleotides primarily through reduced nucleotide binding affinity. Pre-steady-state single-turnover assays on wild-type 9°N polD were used to examine 3'-5' exonuclease hydrolysis activity in the presence of Mg(2+) and Mn(2+). Interestingly, substituting Mn(2+) for Mg(2+) accelerated hydrolysis rates > 40-fold (kexo ≥ 110 s(-1) versus ≥ 2.5 s(-1)). Preference for Mn(2+) over Mg(2+) in exonuclease hydrolysis activity is a property unique to the polD family. The kinetic assays performed in this work provide critical insight into the mechanisms that polD employs to accurately and efficiently replicate the archaeal genome. Furthermore, despite the unique properties of polD, this work suggests that a conserved polymerase kinetic pathway is present in all known DNA polymerase families.
authors.affiliation: From New England Biolabs, Inc., Ipswich, Massachusetts 01938.
authors.forename: Kelly M
authors.initials: KM
authors.lastname: Schermerhorn
authors.affiliation: From New England Biolabs, Inc., Ipswich, Massachusetts 01938 gardner@neb.com.
authors.forename: Andrew F
authors.initials: AF
authors.lastname: Gardner
mesh_terms: D000465:Algorithms; D019843:Archaeal Proteins / Q000235:genetics / Q000378:metabolism*; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism*; D005092:Exonucleases / Q000378:metabolism; D020745:Genome, Archaeal / Q000235:genetics; D006868:Hydrolysis / Q000187:drug effects; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism / Q000494:pharmacology; D008345:Manganese / Q000378:metabolism / Q000494:pharmacology; D009711:Nucleotides / Q000235:genetics / Q000378:metabolism; D019712:Thermococcus / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism
chemical_list: D019843:Archaeal Proteins; D009711:Nucleotides; D008345:Manganese; D004259:DNA-Directed DNA Polymerase; D005092:Exonucleases; D008274:Magnesium
keywords: DNA polymerase; DNA replication; archaea; enzyme kinetics; exonuclease; kinetics; pre-steady-state kinetics