Kinetics of DNA strand transfer between polymerase and proofreading exonuclease active sites regulates error correction during high-fidelity replication.

ABSTRACT: We show that T7 DNA polymerase (pol) and exonuclease (exo) domains contribute to selective error correction during DNA replication by regulating bidirectional strand transfer between the two active sites. To explore the kinetic basis for selective removal of mismatches, we used a fluorescent cytosine analog (1,3-diaza-2-oxophenoxazine) to monitor the kinetics of DNA transfer between the exo and pol sites. We globally fit stopped-flow fluorescence and base excision kinetic data and compared results obtained with ssDNA versus duplex DNA to resolve how DNA transfer governs exo specificity. We performed parallel studies using hydrolysis-resistant phosphorothioate oligonucleotides to monitor DNA transfer to the exo site without hydrolysis. ssDNA binds to the exo site at the diffusion limit (10<

SUBMITTER: Dangerfield TL 

PROVIDER: S-EPMC9800556 | biostudies-literature | 2022 Nov

REPOSITORIES: biostudies-literature