Biochemical basis of DNA replication fidelity.


DNA polymerase is the critical enzyme maintaining genetic integrity ...
DNA polymerase is the critical enzyme maintaining genetic integrity during DNA replication. Individual steps in the replication process that contribute to DNA synthesis fidelity include nucleotide insertion, exonucleolytic proofreading, and binding to and elongation of matched and mismatched primer termini. Each process has been investigated using polyacrylamide gel electrophoresis (PAGE) to resolve 32P-labeled primer molecules extended by polymerase. We describe how integrated gel band intensities can be used to obtain site-specific velocities for addition of correct and incorrect nucleotides, extending mismatched compared to correctly matched primer termini and measuring polymerase dissociation rates and equilibrium DNA binding constants. The analysis is based on steady-state "single completed hit conditions", where polymerases encounter many DNA molecules but where each DNA encounters an enzyme at most once. Specific topics addressed include nucleotide misinsertion, mismatch extension, exonucleolytic proofreading, single nucleotide discrimination using PCR, promiscuous mismatch extension by HIV-1 and AMV reverse transcriptases, sequence context effects on fidelity and polymerase dissociation, structural and kinetic properties of mispairs relating to fidelity, error avoidance mechanisms, kinetics of copying template lesions, the "A-rule" for insertion at abasic template lesions, an interesting exception to the "A-rule", thermodynamic and kinetic determinants of base pair discrimination by polymerases.



Mutational Analysis, Biotech Applications, Kinetic Parameters, Fidelity, Nucleotide Incorporation, Exonuclease Activity, Methods

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Review; many pols touched on.


new topics/pols set partial results complete validated

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