Kinetic analysis of Escherichia coli deoxyribonucleic acid polymerase I.

Travaglini, EC; Mildvan, A S; Loeb, L A

doi:10.1016/s0021-9258(19)40720-5

Cited by 66 publications

(25 citation statements)

References 25 publications

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“…Because the DNA templates we used in this study contain two or more pyrimidine-G-C sites, this may explain why the estimated k cat values are slightly lower than previously estimated rates. Previous measurements of k cat include 8.3, 5.7, 7.5, 7, and 5.3 s –1 . ,,,, Schwartz and Quake calculated a rate as high as 14 s –1 when subtracting pauses. However, there are also k cat values near or below the ones reported here from studies that did not involve pyrimidine-G-C replication sequences such as 3.8, 1.0, and 2.0 s –1 . ,, The estimated K m values of 0.51 μM (Table ) and 0.86 μM (Table ) reported here are in line with previous estimates of 1–2 μM, 0.4–1.7 μM, 1.4 μM, and 0.58 μM …”

Section: Discussionmentioning

confidence: 98%

“…Early experiments with homopolymers found that k cat and K m can differ up to 30fold for different dNTPs. 44 This has, in part, been attributed to the unsuitability of homopolymer templates, because of undesired interactions such as the stacking of T-primers on A-templates. 41 Additional results have, however, confirmed the importance of sequence on polymerization rate: Bertram et al 45 found that both the templating base and neighboring base greatly affect the k cat and K m of dNTP incorporation, while Mytelka and Chamberlin 46 found that T7 DNA polymerase and KF can pause significantly, depending on the environment, during incorporation of dCTP opposite a pyrimidine-G-C site.…”

Section: ■ Discussionmentioning

confidence: 99%

“…Previous measurements of k cat include 8.3, 5.7, 7.5, 7, and 5.3 s −1 . 8,11,37,44,47 Schwartz and Quake 48 calculated a rate as high as 14 s −1 when subtracting pauses. However, there are also k cat values near or below the ones reported here from studies that did not involve pyrimidine-G-C replication sequences such as 3.8, 1.0, and 2.0 s −1 .…”

Section: ■ Discussionmentioning

confidence: 99%

“…Numerous evidence shows that this flexibility would be required for a complete description of processive polymerization to account for the complex dependence of the nucleotide incorporation rate on nucleotide identity (A, C, G, or T) or sequence environment. Early experiments with homopolymers found that k cat and K m can differ up to 30-fold for different dNTPs . This has, in part, been attributed to the unsuitability of homopolymer templates, because of undesired interactions such as the stacking of T-primers on A-templates .…”

Section: Discussionmentioning

confidence: 99%

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Time Course Analysis of Enzyme-Catalyzed DNA Polymerization

2016

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Extracting kinetic parameters from DNA polymerase-catalyzed processive polymerization data using traditional initial-rate analysis has proven to be problematic for multiple reasons. The first substrate, DNA template, is a heterogeneous polymer and binds tightly to DNA polymerase. Further, the affinity and speed of incorporation of the second substrate, deoxynucleoside triphosphate (dNTP), vary greatly depending on the nature of the templating base and surrounding sequence. Here, we present a mathematical model consisting of the DNA template-binding step and a Michaelis-Menten-type nucleotide incorporation step acting on a DNA template with a finite length. The model was numerically integrated and globally fitted to experimental reaction time courses. The time courses were determined by monitoring the processive synthesis of oligonucleotides of lengths between 50 and 120 nucleotides by DNA polymerase I (Klenow fragment exo) using the fluorophore PicoGreen. For processive polymerization, we were able to estimate an enzyme-template association rate k of 7.4 μM s, a disassociation rate k of 0.07 s, and a K of 10 nM, and the steady-state parameters for correct dNTP incorporation give k values of 2.5-3.3 s and K values of 0.51-0.86 μM. From the analysis of time courses measured between 5 and 25 °C, an activation energy for k of 82 kJ mol was calculated, and it was found that up to 73% of Klenow fragment becomes inactivated or involved in unproductive binding at lower temperatures. Finally, a solvent deuterium kinetic isotope effect (KIE) of 3.0-3.2 was observed under processive synthesis conditions, which suggests that either the intrinsic KIE is unusually high, at least 30-40, or previous findings, showing that the phosphoryl transfer step occurs rapidly and is flanked by two slow conformational changes, need to be re-evaluated. We suggest that the numerical integration of rate equations provides a high level of flexibility and generally produces superior results compared to those of initial-rate analysis in the study of DNA polymerase kinetics and, by extension, other complex enzyme systems.

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Section: Discussionmentioning

confidence: 98%

Section: ■ Discussionmentioning

confidence: 99%

Section: ■ Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Time Course Analysis of Enzyme-Catalyzed DNA Polymerization

2016

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“…All NMR solutions, with the exception of those containing either Mg2+ or enzyme were passed through Chelex-100 prior to deuteration to remove trace metal impurities. The concentration of free Mg2+ necessary for optimal activity of whole Pol I was found by Travaglini et al (1975) to range from 0.3 to 2.0 mM, depending on the template used. Repetition of their study with the large fragment of Pol I in 10 mM Tris-HCl, pH 7.50, and 32 mM KC1 using oligo(rU)54±11 as template and dA12_18 as the primer yielded a fairly broad Mg2+ optimum of 1.0-1.7 mM in excess of the deoxynucleoside triphosphate concentration.…”

Section: Methodsmentioning

confidence: 99%

NMR studies of the conformations and interactions of substrates and ribonucleotide templates bound to the large fragment of DNA polymerase I

Ferrin¹,

Mildvan²

1986

Biochemistry

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The large fragment of DNA polymerase I (Pol I) effectively uses oligoribouridylates and oligoriboadenylates as templates, with kinetic properties similar to those of poly(U) and poly(A), respectively, and has little or no activity in degrading them. In the presence of such oligoribonucleotide templates, nuclear Overhauser effects (NOE's) were used to determine interproton distances within and conformations of substrates bound to the large fragment of Pol I, as well as conformations and interactions of the enzyme-bound templates. In the enzyme-oligo(rU)54 +/- 11-Mg2+dATP complex, the substrate dATP has a high anti-glycosidic torsional angle (chi = 62 +/- 10 degrees) and an O1'-endo/C3'-endo sugar pucker (delta = 90 +/- 10 degrees) differing only slightly from those previously found for enzyme-bound dATP in the absence of template [Ferrin, L.J., & Mildvan, A.S. (1985) Biochemistry 24, 4680-4694]. Both conformations are similar to those of deoxynucleotidyl units of B DNA but differ greatly from those of A or Z DNA. The conformation of the enzyme-bound substrate analogue AMPCPP (chi = 50 +/- 10 degrees, delta = 90 +/- 10 degrees) is very similar to that of enzyme-bound dATP and is unaltered by the binding of the template oligo(rU)54 +/- 11 or by the subsequent binding of the primer (Ap)9A. In the enzyme-oligo(rA)50-Mg2+TTP complex, the substrate TTP has an anti-glycosidic torsional angle (chi = 40 +/- 10 degrees) and an O1'-endo sugar pucker (delta = 100 +/- 10 degrees), indistinguishable from those found in the absence of template and compatible with those of B DNA but not with those of A or Z DNA. In the absence of templates, the interproton distances on enzyme-bound dGTP cannot be fit by a single conformation but require a 40% contribution from a syn structure (chi = 222 degrees) and a 60% contribution from one or more anti structures. The presence of the template oligo(rU)43 +/- 9 simplifies the conformation of enzyme-bound dGTP to a single structure with an anti-glycosyl angle (chi = 32 +/- 10 degrees) and an O1'-endo/C3'-endo sugar pucker (delta = 90 +/- 10 degrees), compatible with those of B DNA, possibly due to the formation of a G-U wobble base pair. However, no significant misincorporation of guanine deoxynucleotides by the enzyme is detected with oligo(rU) as template.(ABSTRACT TRUNCATED AT 400 WORDS)

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Incision‐Excision DNA Repair Model: A Theoretical Analysis of the Effect of Enzyme Concentrations on DNA Repair

Bhat

Rao

1989

Ber Bunsenges Phys Chem

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Relationships between the concentration of exonuclease, DNA polymerase, and DNA ligase participating in the DNA incision‐excision repair pathway have been derived by applying steady state conditions to concentration of the species of interest. The analysis utilizes concentrations of damage excised, gapped DNA (DNAm—n) and gap filled unligated DNA (DNAm) at the steady state as the parameters to measure the extent of DNA repair. The concentrations of DNAm—n and DNAm have been expressed in terms of experimentally determinable constants such as Km, catalytic constants for the enzymes, enzyme, and substrate DNA concentrations, making it possible to verify the theoretical predictions. The analysis predicts a low ratio of exonuclease to polymerase to observe any significant repair activity, a conclusion consistent with in vitro observation of Hamilton et al. (1974) that significant DNA repair is observed when exonuclease: polymerase ratio is 0.5. The analyis predicts fairly large concentrations of DNA ligase to minimize DNAm concentration at the steady state. Under specific conditions, reversal of ligation has been predicted and has been suggested as a possible mechanism of initiation of multi‐centered DNA replication in eukaryotes. This suggestion has been substantiated by experimental demonstration that DNA nicked in the presence of DNA ligase and AMP is substrate for M. Luteus DNA polymerase but not for T4 DNA polymerase, suggesting that adenylated DNA intermediate is formed and only cleaved by 5′—3′ exonuclease which is present in vivo, and has been isolated and characterized. The relationships have been numerically evaluated for different enzyme concentrations using derived and literature values for the constants. The potential application of these results for in vitro introduction of “errors” into DNA has been indicated.

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Kinetic analysis of Escherichia coli deoxyribonucleic acid polymerase I.

Cited by 66 publications

References 25 publications

Time Course Analysis of Enzyme-Catalyzed DNA Polymerization

Time Course Analysis of Enzyme-Catalyzed DNA Polymerization

NMR studies of the conformations and interactions of substrates and ribonucleotide templates bound to the large fragment of DNA polymerase I

Incision‐Excision DNA Repair Model: A Theoretical Analysis of the Effect of Enzyme Concentrations on DNA Repair

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