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

2000 ◽  
Author(s):  
Athel Cornish-Bowden
Keyword(s):  
Steady State ◽  
Enzyme Kinetics ◽  
Single Molecule ◽  
Transient State ◽  
Curve Analysis ◽  
Rate Equations ◽  
Progress Curve ◽  
Steady State Kinetics ◽  
Time Regime ◽  
Time Courses

All of chemical kinetics is based on rate equations, but this is especially true of steady-state enzyme kinetics: in other applications a rate equation can be regarded as a differential equation that has to be integrated to give the function of real interest, whereas in steady-state enzyme kinetics it is used as it stands. Although the early enzymologists tried to follow the usual chemical practice of deriving equations that describe the state of reaction as a function of time there were too many complications, such as loss of enzyme activity, effects of accumulating product etc., for this to be a fruitful approach. Rapid progress only became possible when Michaelis and Menten (1) realized that most of the complications could be removed by extrapolating back to zero time and regarding the measured initial rate as the primary observation. Since then, of course, accumulating knowledge has made it possible to study time courses directly, and this has led to two additional subdisciplines of enzyme kinetics, transient-state kinetics, which deals with the time regime before a steady state is established, and progress-curve analysis, which deals with the slow approach to equilibrium during the steady-state phase. The former of these has achieved great importance but is regarded as more specialized. It is dealt with in later chapters of this book. Progress-curve analysis has never recovered the importance that it had at the beginning of the twentieth century. Nearly all steps that form parts of the mechanisms of enzyme-catalysed reactions involve reactions of a single molecule, in which case they typically follow first-order kinetics: . . . v = ka . . . . . . 1 . . . or they involve two molecules (usually but not necessarily different from one another) and typically follow second-order kinetics: . . . v = kab . . . . . . 2 . . . In both cases v represents the rate of reaction, and a and b are the concentrations of the molecules involved, and k is a rate constant. Because we shall be regarding the rate as a quantity in its own right it is not usual in steady-state kinetics to represent it as a derivative such as -da/dt.

scholarly journals Pre-Steady-State Kinetic Analysis of the Incorporation of Anti-HIV Nucleotide Analogs Catalyzed by Human X- and Y-Family DNA Polymerases

2010 ◽  
Vol 55 (1) ◽  
pp. 276-283 ◽  
Author(s):  
Jessica A. Brown ◽  
Lindsey R. Pack ◽  
Jason D. Fowler ◽  
Zucai Suo
Keyword(s):  
Mitochondrial Dna ◽  
Steady State ◽  
Substrate Specificity ◽  
Dna Polymerase ◽  
Dna Polymerases ◽  
Steady State Kinetic ◽  
Y Family ◽  
State Kinetic

ABSTRACTNucleoside reverse transcriptase inhibitors (NRTIs) are an important class of antiviral drugs used to manage infections by human immunodeficiency virus, which causes AIDS. Unfortunately, these drugs cause unwanted side effects, and the molecular basis of NRTI toxicity is not fully understood. Putative routes of NRTI toxicity include the inhibition of human nuclear and mitochondrial DNA polymerases. A strong correlation between mitochondrial toxicity and NRTI incorporation catalyzed by human mitochondrial DNA polymerase has been established bothin vitroandin vivo. However, it remains to be determined whether NRTIs are substrates for the recently discovered human X- and Y-family DNA polymerases, which participate in DNA repair and DNA lesion bypassin vivo. Using pre-steady-state kinetic techniques, we measured the substrate specificity constants for human DNA polymerases β, λ, η, ι, κ, and Rev1 incorporating the active, 5′-phosphorylated forms of tenofovir, lamivudine, emtricitabine, and zidovudine. For the six enzymes, all of the drug analogs were incorporated less efficiently (40- to >110,000-fold) than the corresponding natural nucleotides, usually due to a weaker binding affinity and a slower rate of incorporation for the incoming nucleotide analog. In general, the 5′-triphosphate forms of lamivudine and zidovudine were better substrates than emtricitabine and tenofovir for the six human enzymes, although the substrate specificity profile depended on the DNA polymerase. Our kinetic results suggest NRTI insertion catalyzed by human X- and Y-family DNA polymerases is a potential mechanism of NRTI drug toxicity, and we have established a structure-function relationship for designing improved NRTIs.

Kinetic assays of DNA polymerase fidelity: A theoretical perspective beyond Michaelis-Menten kinetics

2021 ◽  
Vol 104 (1) ◽  
Author(s):  
Qiu-Shi Li ◽  
Yao-Gen Shu ◽  
Zhong-Can Ou-Yang ◽  
Ming Li
Keyword(s):  
Dna Polymerase ◽  
Theoretical Perspective ◽  
Polymerase Fidelity

scholarly journals New in vitro dna polymerase activity and fidelity assay reveals age-dependent changes in Arabidopsis Thaliana

2011 ◽  
Vol 2 (1) ◽  
pp. 7 ◽  
Author(s):  
Andrey Golubov ◽  
Priti Maheshwari ◽  
Andriy Bilichak ◽  
Igor Kovalchuk
Keyword(s):  
Arabidopsis Thaliana ◽  
Dna Repair ◽  
Dna Polymerase ◽  
High Performance ◽  
Dna Polymerases ◽  
Polymerase Activity ◽  
Polymerase Fidelity ◽  
Age Dependent ◽  
Dna Chain

DNA polymerase is an enzyme that adds nucleotides to the growing DNA chain during replication and DNA repair. DNA polymerase activity and fidelity are important characteristics that reflect the ability of DNA polymerase to add nucleotides and then proofread newly synthesized DNA. We have developed a protocol allowing analysis of polymerase activity and fidelity using crude Arabidopsis thaliana plant extracts. It is based on the ability of DNA polymerases in the extract to elongate the fluorescently labelled primer annealed to a short complementary template. For analysis, fluorescently labelled products were separated on a denaturing polyacrylamide gel and visualized using a high performance blot imager. Analysis of tissue prepared from 5-, 12- and 21-day-old Arabidopsis plants showed an age-dependent decrease in polymerase activity, an increase in polymerase fidelity and a tendency to an increase in exo- (endo) nucleolytic activity.

scholarly journals Thioredoxin suppresses microscopic hopping of T7 DNA polymerase on duplex DNA

2010 ◽  
Vol 107 (5) ◽  
pp. 1900-1905 ◽  
Author(s):  
Candice M. Etson ◽  
Samir M. Hamdan ◽  
Charles C. Richardson ◽  
Antoine M. van Oijen
Keyword(s):  
Escherichia Coli ◽  
Dna Replication ◽  
Dna Polymerase ◽  
Single Molecule ◽  
Dna Polymerases ◽  
Model System ◽  
Double Stranded Dna ◽  
Nucleotide Incorporation ◽  
Imaging Approach ◽  
Processivity Factor

The DNA polymerases involved in DNA replication achieve high processivity of nucleotide incorporation by forming a complex with processivity factors. A model system for replicative DNA polymerases, the bacteriophage T7 DNA polymerase (gp5), encoded by gene 5, forms a tight, 1∶1 complex with Escherichia coli thioredoxin. By a mechanism that is not fully understood, thioredoxin acts as a processivity factor and converts gp5 from a distributive polymerase into a highly processive one. We use a single-molecule imaging approach to visualize the interaction of fluorescently labeled T7 DNA polymerase with double-stranded DNA. We have observed T7 gp5, both with and without thioredoxin, binding nonspecifically to double-stranded DNA and diffusing along the duplex. The gp5/thioredoxin complex remains tightly bound to the DNA while diffusing, whereas gp5 without thioredoxin undergoes frequent dissociation from and rebinding to the DNA. These observations suggest that thioredoxin increases the processivity of T7 DNA polymerase by suppressing microscopic hopping on and off the DNA and keeping the complex tightly bound to the duplex.

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.

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

Multi-stage proofreading in DNA replication

1993 ◽  
Vol 26 (3) ◽  
pp. 225-331 ◽  
Author(s):  
Robert A. Beckman ◽  
Lawrence A. Loeb
Keyword(s):  
Steady State ◽  
Dna Replication ◽  
Dna Polymerase ◽  
Dna Polymerases ◽  
Kinetic Studies ◽  
Dna Polymerase I ◽  
E Coli ◽  
Multi Stage ◽  
Base Selection ◽  
Polymerase I

The mechanisms by which DNA polymerases achieve their remarkable fidelity, including base selection and proofreading, are briefly reviewed. Nine proofreading models from the current literature are evaluated in the light of steady-state and transient kinetic studies of E. coli DNA polymerase I, the beststudied DNA polymerase.

PrimPol (Primase/Polymerase), replicating enzyme – A mini review

2020 ◽  
Vol 2 (4) ◽  
pp. 89-92
Author(s):  
Muhammad Amir ◽  
Sabeera Afzal ◽  
Alia Ishaq
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Genetic Information ◽  
Nuclear Dna ◽  
De Novo ◽  
Dna Polymerases ◽  
Test Site ◽  
Mitochondrial Dna Replication ◽  
Dna Template ◽  
Preliminary Phase

Polymerases were revealed first in 1970s. Most important to the modest perception the enzyme responsible for nuclear DNA replication that was pol , for DNA repair pol and for mitochondrial DNA replication pol  DNA construction and renovation done by DNA polymerases, so directing both the constancy and discrepancy of genetic information. Replication of genome initiate with DNA template-dependent fusion of small primers of RNA. This preliminary phase in replication of DNA demarcated as de novo primer synthesis which is catalyzed by specified polymerases known as primases. Sixteen diverse DNA-synthesizing enzymes about human perspective are devoted to replication, reparation, mutilation lenience, and inconsistency of nuclear DNA. But in dissimilarity, merely one DNA polymerase has been called in mitochondria. It has been suggest that PrimPol is extremely acting the roles by re-priming DNA replication in mitochondria to permit an effective and appropriate way replication to be accomplished. Investigations from a numeral of test site have significantly amplified our appreciative of the role, recruitment and regulation of the enzyme during DNA replication. Though, we are simply just start to increase in value the versatile roles that play PrimPol in eukaryote.

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