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Published on: October 6, 2017
RB69 DNA polymerase mutants with expanded nascent base-pair-binding pockets are highly efficient but have reduced
Hong Zhang1, Jeff Beckman, Jimin Wang
1Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520, USA.
Biochemistry
|June 16, 2009
Summary
Enlarging the nascent base-pair-binding pocket of RB69 DNA polymerase increased incorrect nucleotide incorporation. This suggests DNA polymerases use "negative selection" against mispairs rather than solely relying on pocket fit for accuracy.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- DNA polymerases are crucial for DNA replication and repair.
- The nascent base-pair-binding pocket (NBP) plays a key role in DNA polymerase fidelity.
- Understanding NBP's role in discrimination against incorrect nucleotides is essential.
Purpose of the Study:
- To investigate the effect of enlarging the NBP of bacteriophage RB69 DNA polymerase (RB69 pol) on nucleotide incorporation efficiency.
- To determine how modifications to specific NBP residues impact the polymerase's ability to incorporate correct and incorrect deoxynucleoside monophosphates (dNMPs).
Main Methods:
- Systematic mutagenesis of RB69 pol by replacing residues L561, Y567, and S565 in the NBP with alanine (Ala) and glycine (Gly).
- Construction of single, double, and triple mutants to progressively enlarge the NBP.
- Kinetic analysis of dNMP incorporation efficiency (k(pol)/K(d,app)) for both correct and incorrect dNMPs using wild-type and mutant RB69 pol.
Main Results:
- Enlarging the NBP significantly increased the incorporation efficiency of incorrect dNMPs, with effects escalating with the number of mutations.
- The increased misincorporation was primarily due to changes in the catalytic rate (k(pol)).
- Surprisingly, enlarging the NBP had minimal impact on the incorporation efficiency of correct dNMPs.
Conclusions:
- Replicative DNA polymerases like RB69 pol likely employ "negative selection" against mispairs via NBP residues, rather than solely relying on a tight fit, to ensure accuracy.
- This mechanism allows for the rapid incorporation of correct base pairs while disfavoring incorrect ones.
- Findings contrast with models emphasizing NBP 'tightness of fit' and align with studies on translesion polymerases regarding substrate fit in catalytic centers.
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