DNA polymerase proofreading: active site switching catalyzed by the bacteriophage T4 DNA polymerase.
DNA polymerases achieve high-fidelity DNA replication in part by checking the accuracy of each nucleotide that is incorporated and, if a mistake is made, the incorrect nucleotide is removed before further primer extension takes place. In order to proofread, the primer-end must be separated from the template strand and transferred from the polymerase to the exonuclease active center where the excision reaction takes place; then the trimmed primer-end is returned to the polymerase active center. Thus, proofreading requires polymerase-to-exonuclease and exonuclease-to-polymerase active site switching. We have used a fluorescence assay that uses differences in the fluorescence intensity of 2-aminopurine (2AP) to measure the rates of active site switching for the bacteriophage T4 DNA polymerase. There are three findings: (i) the rate of return of the trimmed primer-end from the exonuclease to the polymerase active center is rapid, >500 s(-1); (ii) T4 DNA polymerase can remove two incorrect nucleotides under single turnover conditions, which includes presumed exonuclease-to-polymerase and polymerase-to-exonuclease active site switching steps and (iii) proofreading reactions that initiate in the polymerase active center are not intrinsically processive.
Fidalgo da Silva
Elizabeth
E
Department of Biological Sciences, University of Alberta, Edmonton, Alberta T6G 2E9, Canada.
Reha-Krantz
Linda J
LJ
D015075:2-Aminopurine / Q000737:chemistry; D019943:Amino Acid Substitution; D020137:Base Pair Mismatch; D001665:Binding Sites; D002384:Catalysis; D004247:DNA / Q000096:biosynthesis / Q000737:chemistry; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism*; D005090:Exodeoxyribonucleases / Q000378:metabolism*; D005453:Fluorescence; D007700:Kinetics; D009711:Nucleotides / Q000378:metabolism; D013941:Thymine / Q000737:chemistry; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
D009711:Nucleotides; D014764:Viral Proteins; C069983:gene 43 protein, Enterobacteria phage T4; D015075:2-Aminopurine; D004247:DNA; D004259:DNA-Directed DNA Polymerase; D005090:Exodeoxyribonucleases; D013941:Thymine
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