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Probing DNA Base-Dependent Leaving Group Kinetic Effects on the DNA Polymerase Transition State.

ABSTRACT: We examine the DNA polymerase ? (pol ?) transition state (TS) from a leaving group pre-steady-state kinetics perspective by measuring the rate of incorporation of dNTPs and corresponding novel ?,?-CXY-dNTP analogues, including individual ?,?-CHF and -CHCl diastereomers with defined stereochemistry at the bridging carbon, during the formation of right (R) and wrong (W) base pairs. Brønsted plots of log kpol versus p Ka4 of the leaving group bisphosphonic acids are used to interrogate the effects of the base identity, the dNTP analogue leaving group basicity, and the precise configuration of the C-X atom in R and S stereoisomers on the rate-determining step ( kpol). The dNTP analogues provide a range of leaving group basicity and steric properties by virtue of monohalogen, dihalogen, or methyl substitution at the carbon atom bridging the ?,?-bisphosphonate that mimics the natural pyrophosphate leaving group in dNTPs. Brønsted plot relationships with negative slopes are revealed by the data, as was found for the dGTP and dTTP analogues, consistent with a bond-breaking component to the TS energy. However, greater multiplicity was shown in the linear free energy relationship, revealing an unexpected dependence on the nucleotide base for both A and C. Strong base-dependent perturbations that modulate TS relative to ground-state energies are likely to arise from electrostatic effects on catalysis in the pol active site. Deviations from a uniform linear Brønsted plot relationship are discussed in terms of insights gained from structural features of the prechemistry DNA polymerase active site.

SUBMITTER: Oertell K

PROVIDER: S-EPMC6039227 | biostudies-literature | 2018 Jul

REPOSITORIES: biostudies-literature

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Probing DNA Base-Dependent Leaving Group Kinetic Effects on the DNA Polymerase Transition State.

Oertell Keriann K Kashemirov Boris A BA Negahbani Amirsoheil A Minard Corinne C Haratipour Pouya P Alnajjar Khadijeh S KS Sweasy Joann B JB Batra Vinod K VK Beard William A WA Wilson Samuel H SH McKenna Charles E CE Goodman Myron F MF

Biochemistry 20180619 26

We examine the DNA polymerase β (pol β) transition state (TS) from a leaving group pre-steady-state kinetics perspective by measuring the rate of incorporation of dNTPs and corresponding novel β,γ-CXY-dNTP analogues, including individual β,γ-CHF and -CHCl diastereomers with defined stereochemistry at the bridging carbon, during the formation of right (R) and wrong (W) base pairs. Brønsted plots of log k<sub>pol</sub> versus p K<sub>a4</sub> of the leaving group bisphosphonic acids are used to in ...[more]

PMID: 29889506

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Project description:BACKGROUND:Transmission of Plasmodium greatly depends on the foraging behaviour of its mosquito vector (Anopheles spp.). The accessibility of blood hosts and availability of plant sugar (i.e., nectar) sources, together with mosquito energy state, have been shown to modulate blood feeding (and thus biting rates) of anopheline mosquitoes. In this study, the influence of mosquito starvation status and availability of nectar on the decision of female Anopheles gambiae mosquitoes to leave a bed net-protected blood host was examined. METHODS:Two small-scale mesocosm experiments were conducted using female mosquitoes starved for 0, 24 or 48 h, that were released inside a specially constructed hut with mesh-sealed exits and containing a bed net-protected human volunteer. Floral cues were positioned on one side of the hut or the other. Several biologically plausible exponential decay models were developed that characterized the emigration rates of mosquitoes from the huts. These varied from simple random loss to leaving rates dependent upon energy state and time. These model fits were evaluated by examining their fitted parameter estimates and comparing Akaike information criterion. RESULTS:Starved mosquitoes left domiciles at a higher rate than recently fed individuals however, there was no difference between 1- and 2-day-starved mosquitoes. There was also no effect of floral cue placement. The best fitting emigration model was one based on both mosquito energy state and time whereas the worst fitting model was one based on the assumption of constant leaving rates, independent of time and energy state. CONCLUSIONS:The results confirm that mosquito-leaving behaviour is energy-state dependent, and provide some of the first evidence of state-dependent domicile emigration in An. gambiae, which may play a role in malarial transmission dynamics. Employment of simple, first-principle, mechanistic models can be very useful to our understanding of why and how mosquitoes leave domiciles.

Dataset Information

Probing DNA Base-Dependent Leaving Group Kinetic Effects on the DNA Polymerase Transition State.

Publications

Probing DNA Base-Dependent Leaving Group Kinetic Effects on the DNA Polymerase Transition State.

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