Local conformations and competitive binding affinities of single- and double-stranded primer-template DNA at the polymerization and editing active sites of DNA polymerases.
In addition to their capacity for template-directed 5' --> 3' DNA synthesis at the polymerase (pol) site, DNA polymerases have a separate 3' --> 5' exonuclease (exo) editing activity that is involved in assuring the fidelity of DNA replication. Upon misincorporation of an incorrect nucleotide residue, the 3' terminus of the primer strand at the primer-template (P/T) junction is preferentially transferred to the exo site, where the faulty residue is excised, allowing the shortened primer to rebind to the template strand at the pol site and incorporate the correct dNTP. Here we describe the conformational changes that occur in the primer strand as it shuttles between the pol and exo sites of replication-competent Klenow and Klentaq DNA polymerase complexes in solution and use these conformational changes to measure the equilibrium distribution of the primer between these sites for P/T DNA constructs carrying both matched and mismatched primer termini. To this end, we have measured the fluorescence and circular dichroism spectra at wavelengths of >300 nm for conformational probes comprising pairs of 2-aminopurine bases site-specifically replacing adenine bases at various positions in the primer strand of P/T DNA constructs bound to DNA polymerases. Control experiments that compare primer conformations with available x-ray structures confirm the validity of this approach. These distributions and the conformational changes in the P/T DNA that occur during template-directed DNA synthesis in solution illuminate some of the mechanisms used by DNA polymerases to assure the fidelity of DNA synthesis.
Datta
Kausiki
K
From the Institute of Molecular Biology and Department of Chemistry, University of Oregon, Eugene, Oregon 97403-1229.
Johnson
Neil P
NP
Institut de Pharmacologie et de Biologie Structurale, UMR 5089, CNRS, 205 Route de Narbonne, 31077 Toulouse, France.
LiCata
Vince J
VJ
Department of Biological Sciences, Louisiana State University, Baton Rouge, Louisiana 70803.
von Hippel
Peter H
PH
From the Institute of Molecular Biology and Department of Chemistry, University of Oregon, Eugene, Oregon 97403-1229. Electronic address: petevh@molbio.uoregon.edu.
D001483:Base Sequence; D001667:Binding, Competitive; D002118:Calcium / Q000378:metabolism; D020134:Catalytic Domain; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D004247:DNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004256:DNA Polymerase I / Q000737:chemistry / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000378:metabolism*; D046911:Macromolecular Substances; D008274:Magnesium / Q000378:metabolism; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D011487:Protein Conformation
D017931:DNA Primers; D004277:DNA, Single-Stranded; D046911:Macromolecular Substances; D004247:DNA; D004256:DNA Polymerase I; D004259:DNA-Directed DNA Polymerase; D008274:Magnesium; D002118:Calcium