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Elucidation of kinetic mechanisms of human translesion DNA polymerase ? using tryptophan mutants.


ABSTRACT: To investigate the conformational dynamics of human DNA polymerase ? (hpol ?), we generated two mutants, Y50W (N-clasp region) and Y408W (linker between the thumb and little finger domains), using a Trp-null mutant (W214Y/W392H) of the hpol ? catalytic core enzyme. These mutants retained catalytic activity and similar patterns of selectivity for bypassing the DNA adduct 7,8-dihydro-8-oxo-2'-deoxyguanosine, as indicated by the results of steady-state and pre-steady-state kinetic experiments. Stopped-flow kinetic assays with hpol ? Y50W and T408W revealed a decrease in Trp fluorescence with the template G:dCTP pair but not for any mispairs. This decrease in fluorescence was not rate-limiting and is considered to be related to a conformational change necessary for correct nucleotidyl transfer. When a free 3'-hydroxyl was present on the primer, the Trp fluorescence returned to the baseline level at a rate similar to the observed kcat , suggesting that this change occurs during or after nucleotidyl transfer. However, polymerization rates (kpol ) of extended-product formation were fast, indicating that the slow fluorescence step follows phosphodiester bond formation and is rate-limiting. Pyrophosphate formation and release were fast and are likely to precede the slower relaxation step. The available kinetic data were used to fit a simplified minimal model. The extracted rate constants confirmed that the conformational change after phosphodiester bond formation was rate-limiting for hpol ? catalysis with the template G:dCTP pair.

SUBMITTER: Zhao L 

PROVIDER: S-EPMC4182141 | biostudies-literature | 2014 Oct

REPOSITORIES: biostudies-literature

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Elucidation of kinetic mechanisms of human translesion DNA polymerase κ using tryptophan mutants.

Zhao Linlin L   Pence Matthew G MG   Eoff Robert L RL   Yuan Shuai S   Fercu Catinca A CA   Guengerich F Peter FP  

The FEBS journal 20140814 19


To investigate the conformational dynamics of human DNA polymerase κ (hpol κ), we generated two mutants, Y50W (N-clasp region) and Y408W (linker between the thumb and little finger domains), using a Trp-null mutant (W214Y/W392H) of the hpol κ catalytic core enzyme. These mutants retained catalytic activity and similar patterns of selectivity for bypassing the DNA adduct 7,8-dihydro-8-oxo-2'-deoxyguanosine, as indicated by the results of steady-state and pre-steady-state kinetic experiments. Stop  ...[more]

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