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Manganese substantially alters the dynamics of translesion DNA synthesis
Heather Hays1, Anthony J Berdis
1Department of Chemistry, Case Western Reserve University, 2109 Adelbert Road, Cleveland, Ohio 44106, USA.
Abstract:
The effect of metal ion substitution on the dynamics of translesion DNA synthesis catalyzed by the bacteriophage T4 DNA polymerase was quantitatively evaluated through steady-state and transient kinetic techniques. Substitution of Mn(2+) for Mg(2+) enhances the steady-state rate of dNMP misinsertion opposite an abasic site by 11-34-fold. At the molecular level, the enhancement in translesion DNA synthesis reflects a substantial increase in the rate of the conformational change preceding phosphoryl transfer for all dNTPs that were tested. This is best illustrated by the biphasic pre-steady-state time course of dAMP insertion opposite an abasic site which indicates that a step after chemistry is rate-limiting for steady-state enzyme turnover. Furthermore, the k(pol) value of 40 s(-1) measured under single-turnover reaction conditions is 20-fold greater than the k(cat) value of 2 s(-1) measured for steady-state enzyme turnover. Finally, the low elemental effect ( approximately 2.4-fold reduction in k(pol)) measured by substituting the alpha-thiotriphosphate analogue for dATP further argues that chemistry is not rate-limiting. In contrast to the biphasic insertion of dAMP, pre-steady-state time courses for the insertion of dCMP, dGMP, or dTMP opposite an abasic site were linear. Nearly identical k(pol) values ( approximately 1 s(-1)) were measured for the insertion of dCMP, dGMP, and dTMP opposite the abasic site using single-turnover conditions. However, the large elemental effects of 27 and 70 measured by substituting the alpha-thiotriphosphate analogues for dCTP and dGTP, respectively, suggest that phosphoryl transfer may be the rate-limiting step for their insertion opposite the abasic site. Various models are discussed in an attempt to explain the effect of metal ion substitution on the dynamics of translesion DNA replication.
Insights
Manganese ions significantly boost T4 DNA polymerase
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Translesion DNA synthesis is crucial for genome stability, allowing DNA polymerases to bypass damaged DNA bases.
- Bacteriophage T4 DNA polymerase is a model system for studying DNA replication fidelity and repair mechanisms.
- Metal ions, particularly Mg(2+) and Mn(2+), are essential cofactors for DNA polymerase activity.
Purpose of the Study:
- To investigate the impact of substituting Mn(2+) for Mg(2+) on the kinetic mechanism of T4 DNA polymerase during translesion synthesis.
- To elucidate the rate-limiting steps in nucleotide insertion opposite an abasic site under different metal ion conditions.
Main Methods:
- Quantitative evaluation using steady-state and transient kinetic techniques.
- Measurement of enzyme kinetics, including rate constants (k(pol), k(cat)) and elemental effects.
- Analysis of DNA polymerase activity with various deoxynucleoside triphosphates (dNTPs) opposite an abasic site.
Main Results:
- Mn(2+) substitution enhanced the misinsertion rate of dNMP opposite an abasic site by 11-34 fold compared to Mg(2+).
- Mn(2+) accelerated the conformational change preceding phosphoryl transfer, with a rate-limiting step after chemistry for dAMP insertion.
- Phosphoryl transfer appeared rate-limiting for dCMP and dGMP insertion under Mn(2+) conditions, indicated by large elemental effects.
Conclusions:
- Metal ion substitution significantly alters the dynamics of translesion DNA synthesis by T4 DNA polymerase.
- Mn(2+) promotes faster conformational changes but can make phosphoryl transfer rate-limiting for certain nucleotides.
- Understanding these kinetic effects provides insights into DNA repair fidelity and polymerase mechanism.