title: Modulation of DNA Polymerase Noncovalent Kinetic Transitions by Divalent Cations.
abstract: Replicative DNA polymerases (DNAPs) require divalent metal cations for phosphodiester bond formation in the polymerase site and for hydrolytic editing in the exonuclease site. Me(2+) ions are intimate architectural components of each active site, where they are coordinated by a conserved set of amino acids and functional groups of the reaction substrates. Therefore Me(2+) ions can influence the noncovalent transitions that occur during each nucleotide addition cycle. Using a nanopore, transitions in individual Φ29 DNAP complexes are resolved with single-nucleotide spatial precision and sub-millisecond temporal resolution. We studied Mg(2+) and Mn(2+), which support catalysis, and Ca(2+), which supports deoxynucleoside triphosphate (dNTP) binding but not catalysis. We examined their effects on translocation, dNTP binding, and primer strand transfer between the polymerase and exonuclease sites. All three metals cause a concentration-dependent shift in the translocation equilibrium, predominantly by decreasing the forward translocation rate. Me(2+) also promotes an increase in the backward translocation rate that is dependent upon the primer terminal 3'-OH group. Me(2+) modulates the translocation rates but not their response to force, suggesting that Me(2+) does not affect the distance to the transition state of translocation. Absent Me(2+), the primer strand transfer pathway between the polymerase and exonuclease sites displays additional kinetic states not observed at >1 mm Me(2+). Complementary dNTP binding is affected by Me(2+) identity, with Ca(2+) affording the highest affinity, followed by Mn(2+), and then Mg(2+). Both Ca(2+) and Mn(2+) substantially decrease the dNTP dissociation rate relative to Mg(2+), while Ca(2+) also increases the dNTP association rate.
authors.affiliation: From the Departments of Biomolecular Engineering and.
authors.forename: Joseph M
authors.initials: JM
authors.lastname: Dahl
authors.affiliation: From the Departments of Biomolecular Engineering and krlieberman@gmail.com.
authors.forename: Kate R
authors.initials: KR
authors.lastname: Lieberman
authors.affiliation: Applied Mathematics and Statistics, University of California, Santa Cruz, California 95064 hongwang@soe.ucsc.edu.
authors.forename: Hongyun
authors.initials: H
authors.lastname: Wang
mesh_terms: D019943:Amino Acid Substitution; D001435:Bacteriophages / Q000201:enzymology; D055162:Biocatalysis; D002122:Calcium Chloride / Q000737:chemistry*; D002712:Chlorides / Q000737:chemistry*; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry*; D003843:Deoxycytidine Monophosphate / Q000737:chemistry; D003848:Deoxyguanine Nucleotides / Q000737:chemistry; D007700:Kinetics; D015636:Magnesium Chloride / Q000737:chemistry*; D017895:Manganese Compounds / Q000737:chemistry*; D058105:Polymerization; D011485:Protein Binding; D014764:Viral Proteins / Q000737:chemistry*
chemical_list: D002712:Chlorides; D003848:Deoxyguanine Nucleotides; D017895:Manganese Compounds; D014764:Viral Proteins; D015636:Magnesium Chloride; D003843:Deoxycytidine Monophosphate; C029603:deoxyguanosine triphosphate; D004259:DNA-Directed DNA Polymerase; D002122:Calcium Chloride; C025340:manganese chloride
keywords: DNA polymerase; DNA replication; enzyme kinetics; enzyme mechanism; single-molecule biophysics