scholarly journals DNA Polymerase Fidelity: Comparing Direct Competition of Right and Wrong dNTP Substrates with Steady State and Pre-Steady State Kinetics

Biochemistry ◽  
2010 ◽  
Vol 49 (1) ◽  
pp. 20-28 ◽  
Author(s):  
Jeffrey G. Bertram ◽  
Keriann Oertell ◽  
John Petruska ◽  
Myron F. Goodman
Keyword(s):  
Steady State ◽  
Dna Polymerase ◽  
Polymerase Fidelity ◽  
Steady State Kinetics ◽  
Direct Competition

scholarly journals DNA polymerase β: analysis of the contributions of tyrosine-271 and asparagine-279 to substrate specificity and fidelity of DNA replication by pre-steady-state kinetics

1997 ◽  
Vol 323 (1) ◽  
pp. 103-111 ◽  
Author(s):  
Vadim S. KRAYNOV ◽  
Brian G. WERNEBURG ◽  
Xuejun ZHONG ◽  
Hui LEE ◽  
Jinwoo AHN ◽  
...  
Keyword(s):  
Steady State ◽  
Substrate Specificity ◽  
Dna Polymerase ◽  
Excision Repair ◽  
Catalytic Efficiency ◽  
Main Function ◽  
Wild Type ◽  
Active Site Residues ◽  
Dna Polymerase Β ◽  
Steady State Kinetics

DNA polymerase β (pol β) from rat brain, overexpressed in Escherichia coli, was used as a model to study the factors responsible for substrate specificity [kpol, Kd(app) and kpol/Kd (app)] and fidelity during DNA synthesis. The roles of two active-site residues, Asn-279 and Tyr-271, were examined by construction of N279A, N279Q, Y271A, Y271F and Y271S mutants followed by structural analyses by NMR and CD and functional analyses by pre-steady-state kinetics. The results are summarized as follows. (i) None of the two-dimensional NMR spectra of the mutants was significantly perturbed relative to that for wild-type pol β, suggesting that Tyr-271 and Asn-279 are not important for the global structure of the protein. (ii) CD analyses of guanidinium hydrochloride-induced denaturation showed that all mutants behaved similarly to the wild type in the free energy of denaturation, suggesting that Tyr-271 and Asn-279 are not critical for the conformational stability of pol β. (iii) The Kd(app) for the correct dNTP was lower than that for the incorrect dNTP by a factor of 10-30 in the case of wild-type pol β. Upon mutation to give N279A and N279Q, the Kd(app) for the correct dNTP increased by a factor of 15-25. As a consequence, the Kd(app) values for the correct and incorrect nucleotides were similar for N279A and N279Q, suggesting that the main function of the side chain of Asn-279 is in discrimination between the binding of correct and incorrect dNTPs. (iv) In the case of the Y271A mutant, the fidelity and the catalytic efficiency kpol/Kd(app) were little perturbed relative to the wild type. However, both the kpol and Kd(app) values for dNTP were 4-8 times lower in the case of the Y271A mutant than the corresponding values for wild-type pol β. Since the chemical step may not be rate-limiting for wild-type pol β, the effect on kpol could be quite significant if it is caused by a perturbation in the chemical step. (v) Pol β displayed the greatest specificity towards the G:C base pair, which is incorporated during base excision repair of G:U and G:T mispairs. This specificity was slightly enhanced for the Y271F mutant.

scholarly journals A critical survey on the kinetic assays of DNA polymerase fidelity from a new theoretical perspective

2020 ◽  
Author(s):  
Qiu-Shi Li ◽  
Yao-Gen Shu ◽  
Zhong-Can Ou-Yang ◽  
Ming Li
Keyword(s):  
Steady State ◽  
Dna Polymerase ◽  
Single Molecule ◽  
Theoretical Perspective ◽  
Dna Polymerases ◽  
Transient State ◽  
Critical Survey ◽  
Polymerase Fidelity ◽  
Error Frequency ◽  
Kinetic Rates

The high fidelity of DNA polymerase is critical for the faithful replication of genomic DNA. Several approaches were proposed to quantify the fidelity of DNA polymerase. Direct measurements of the error frequency of the replication products definitely give the true fidelity but turn out very hard to implement. Two biochemical kinetic approaches, the steady-state assay and the transient-state assay, were then suggested and widely adopted. In these assays, the error frequency is indirectly estimated by using the steady-state or the transient-state kinetic theory combined with the measured kinetic rates. However, whether these indirectly estimated fidelities are equivalent to the true fidelity has never been clarified theoretically, and in particular there are different strategies to quantify the proofreading efficiency of DNAP but often lead to inconsistent results. The reason for all these confusions is that it’s mathematically challenging to formulate a rigorous and general theory of the true fidelity. Recently we have succeeded to establish such a theoretical framework. In this paper, we develop this theory to make a comprehensive examination on the theoretical foundation of the kinetic assays and the relation between fidelities obtained by different methods. We conclude that while the steady-state assay and the transient-state assay can always measure the true fidelity of exonuclease-deficient DNA polymerases, they only do so for exonuclease-efficient DNA polymerases conditionally (the proper way to use these assays to quantify the proofreading efficiency is also suggested). We thus propose a new kinetic approach, the single-molecule assay, which indirectly but precisely characterizes the true fidelity of either exonuclease-deficient or exonuclease-efficient DNA polymerases.

Steady-state kinetics of mouse DNA polymerase .beta.

Biochemistry ◽  
1979 ◽  
Vol 18 (15) ◽  
pp. 3401-3406 ◽  
Author(s):  
K. Tanabe ◽  
E. W. Bohn ◽  
S. H. Wilson
Keyword(s):  
Steady State ◽  
Dna Polymerase ◽  
Dna Polymerase Beta ◽  
Polymerase Beta ◽  
Steady State Kinetics ◽  
Kinetics Of

scholarly journals Mechanism of Efficient and Accurate Nucleotide Incorporation Opposite 7,8-Dihydro-8-Oxoguanine by Saccharomyces cerevisiae DNA Polymerase η

2005 ◽  
Vol 25 (6) ◽  
pp. 2169-2176 ◽  
Author(s):  
Karissa D. Carlson ◽  
M. Todd Washington
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Steady State ◽  
Binding Affinity ◽  
Dna Polymerase ◽  
Dna Polymerases ◽  
Lower Efficiency ◽  
Steady State Kinetics ◽  
Nucleotide Incorporation ◽  
Unique Ability

ABSTRACT Most DNA polymerases incorporate nucleotides opposite template 7,8-dihydro-8-oxoguanine (8-oxoG) lesions with reduced efficiency and accuracy. DNA polymerase (Pol) η, which catalyzes the error-free replication of template thymine-thymine (TT) dimers, has the unique ability to accurately and efficiently incorporate nucleotides opposite 8-oxoG templates. Here we have used pre-steady-state kinetics to examine the mechanisms of correct and incorrect nucleotide incorporation opposite G and 8-oxoG by Saccharomyces cerevisiae Pol η. We found that Pol η binds the incoming correct dCTP opposite both G and 8-oxoG with similar affinities, and it incorporates the correct nucleotide bound opposite both G and 8-oxoG with similar rates. While Pol η incorporates an incorrect A opposite 8-oxoG with lower efficiency than it incorporates a correct C, it does incorporate A more efficiently opposite 8-oxoG than opposite G. This is mainly due to greater binding affinity for the incorrect incoming dATP opposite 8-oxoG. Overall, these results show that Pol η replicates through 8-oxoG without any barriers introduced by the presence of the lesion.

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