Elucidation of kinetic mechanisms of human translesionDNApolymerase κ using tryptophan mutants

Abstract

In order 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 (8-oxoG), as judged by the results of steady-state and pre-steady-state kinetic experiments. Stopped-flow kinetic assays with hpol κ 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 formation was rate-limiting for hpol κ catalysis with the template G:dCTP pair.

Human DNA Polymerase ν Catalyzes Correct and Incorrect DNA Synthesis with High Catalytic Efficiency

“…This value is larger than the k pol found for the Y-family polymerases and but similar to that of pol (30,38,39).…”

“…When an incorrect dNTP binds, the enzyme is unable to adopt the highly catalytic conformation, and phosphodiester bond formation becomes rate-limiting (11,12,18,28). The kinetic schemes of the low fidelity Dpo4 and pol also consist of those seven steps, with the difference that the rate phosphodiester bond formation is similar to those of the conformational changes (29,30). In this study, we evaluated the kinetic mechanism of pol and found that similar to KF and T7 pol, pol catalyzes correct base pair formation with a rapid phosphodiester bond formation step.…”

Kinetic and Structural Mechanisms of (5′S)-8,5′-Cyclo-2′-deoxyguanosine-Induced DNA Replication Stalling

“…This decrease, deriving from a 4-fold decrease in k pol and a 27-fold increase in K d,dCTP,app for the S-cdG:dCTP pair compared to those of the dG:dCTP pair, suggests that S-cdG attenuated nucleotide incorporation due to weakened nucleotide binding and a lowered k pol [function of the rate of the forward conformational change and the chemistry step (see Discussion)]. 38 The Dpo4-catalyzed dCTP incorporation opposite S-cdG (Table 3 and Figure S2E,F of the Supporting Information) was somewhat unusual in that its k pol was independent of the dCTP concentration ( Figure S2F of the Supporting Information, inset). Instead, the product amplitude was dependent on the dCTP concentration ( Figure S2F of the Supporting Information), which suggests that the polymerization is readily reversible, so that the increase in dCTP concentration drives an increasing amount of product formed at the enzyme active site.…”

“…Pre-steady-state kinetics is powerful in dissecting the catalytic mechanisms of an enzyme. 38 We used pre-steady-state burst kinetics (or active site titration) to examine the transient concentration of the productive pol:DNA:dNTP complex during catalysis ( Figure 4 and Table 2). This concentration is important for defining the active portion of the enzyme in a certain protein preparation, and it is also meaningful in explaining the partition between the productive and nonproductive complexes during TLS.…”

“…38 Pre-Steady-State Kinetics. Pre-steady-state kinetic assays were performed on a KinTek RQF-3 model chemical quenchflow apparatus (KinTek Corp., Austin, TX).…”