Studies on the affinity of chick embryo DNA ligase for ribonucleotides and deoxyribonucleotides

David, Jean‐Claude

doi:10.1016/0014-5793(77)81078-8

Cited by 5 publications

(3 citation statements)

References 10 publications

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“…Cooper and Rudolph [10], discussing the results of Teraoka et al [8], pointed out that ordered release of sealed DNA then AMP would prevent product inhibition by sealed DNA within cells as long as the AMP concentration is low relative to its K I . Other investigators have measured the potency of inhibition of DNA ligases by AMP [11,12] or its binding affinity [13]. In no case, however, has the steady-state rate equation including product concentrations been published for the Bi Ter Ping Pong kinetic mechanism of DNA ligase.…”

Section: Resultsmentioning

confidence: 99%

Complete steady-state rate equation for DNA ligase and its use for measuring product kinetic parameters of NAD+-dependent DNA ligase from Haemophilus influenzae

Shapiro

2014

BMC Res Notes

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BackgroundDNA ligase seals the nicks in the phosphodiester backbone between Okazaki fragments during DNA replication. DNA ligase has an unusual Bi Ter Ping Pong kinetic mechanism. Its substrates in eubacteria are NAD+ and nicked DNA (nDNA). Its products are nicotinamide mononucleotide (NMN), adenosine 5′-monophosphate (AMP), and sealed DNA. Investigation of the kinetic mechanism and measurement of the kinetic constants of DNA ligase using steady-state kinetics would benefit from the availability of the complete steady-state rate equation, including terms for product concentrations and product-related kinetic constants, which has not previously been published.ResultsThe rate equations for two possible Bi Ter kinetic mechanisms for DNA ligase, including products, are reported. The mechanisms differ according to whether the last two products, AMP and sealed DNA, are released in an ordered or rapid-equilibrium random (RER) manner. Steady-state kinetic studies of product inhibition by NMN and AMP were performed with Haemophilus influenzae NAD+-dependent DNA ligase. The complete rate equation enabled measurement of dissociation constants for NAD+, NMN, and AMP and eliminated one of 3 possible product release mechanisms.ConclusionsSteady-state kinetic product inhibition experiments and complete steady-state kinetic rate equations were used to measure dissociation constants of NAD+, NMN, and AMP and eliminate the possibility that AMP is the second product released in an ordered mechanism. Determining by steady-state kinetics whether the release of sealed DNA and AMP products goes by an ordered (AMP last off) or RER mechanism was shown to require a product inhibition study using sealed DNA.

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Section: Resultsmentioning

confidence: 99%

Complete steady-state rate equation for DNA ligase and its use for measuring product kinetic parameters of NAD+-dependent DNA ligase from Haemophilus influenzae

Shapiro

2014

BMC Res Notes

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“…The inhibitory effect of 5-FU on HepG2 was determined by the cytotoxic MTT assay. HepG2 cells were seeded in 96 wells plate (LabServ, Thermo Fisher Scientific Co., Beijing, China) at 1 × 10 4 cells/well. After incubation, they were treated with the 5-FU at different concentrations for 72 h. MTT solution (final concentration of 0.5 mg/ml in medium) was added to each well and incubated further for 4 h. The medium was removed, and 100 μl of DMSO was added to each well to dissolve the purple crystals of formazan.…”

Section: Methodsmentioning

confidence: 99%

“…The ribonucleotides (RN) and deoxyribonucleotides (dRN) are essential metabolites that play important roles in a broad range of key cellular functions such as DNA synthesis and repair as well as energy metabolism 1 2 3 4 . Deoxyribouncleoside monophosphates (dNMP), deoxyribouncleoside diphosphates (dNDP) and deoxyribouncleoside triphosphates (dNTP) are major metabolites of dRN metabolism and building blocks of DNA synthesis 5 .…”

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confidence: 99%

Application of artificial neural network to investigate the effects of 5-fluorouracil on ribonucleotides and deoxyribonucleotides in HepG2 cells

Guo

Chen

Lam

et al. 2015

Sci Rep

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Endogenous ribonucleotides and deoxyribonucleotides are essential metabolites that play important roles in a broad range of key cellular functions. Their intracellular levels could also reflect the action of nucleoside analogues. We investigated the effects of 5-fluorouracil (5-FU) on ribonucleotide and deoxyribonucleotide pool sizes in cells upon exposure to 5-FU for different durations. Unsupervised and supervised artificial neural networks were compared for comprehensive analysis of global responses to 5-FU. As expected, deoxyuridine monophosphate (dUMP) increased after 5-FU incubation due to the inhibition of thymine monophosphate (TMP) synthesis. Interestingly, the accumulation of dUMP could not lead to increased levels of deoxyuridine triphosphate (dUTP) and deoxyuridine diphosphate (dUDP). After the initial fall in intracellular deoxythymidine triphosphate (TTP) concentration, its level recovered and increased from 48 h exposure to 5-FU, although deoxythymidine diphosphate (TDP) and TMP continued to decrease compared with the control group. These findings suggest 5-FU treatment caused unexpected changes in intracellular purine polls, such as increases in deoxyadenosine triphosphate (dATP), adenosine-triphosphate (ATP), guanosine triphosphate (GTP) pools. Further elucidation of the mechanism of action of 5-FU in causing these changes should enhance development of strategies that will increase the anticancer activity of 5-FU while decreasing its resistance.

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Affinity chromatography in DNA ligase purification

David¹

1979

Advanced Technobiology

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Studies on the affinity of chick embryo DNA ligase for ribonucleotides and deoxyribonucleotides

Cited by 5 publications

References 10 publications

Complete steady-state rate equation for DNA ligase and its use for measuring product kinetic parameters of NAD+-dependent DNA ligase from Haemophilus influenzae

Complete steady-state rate equation for DNA ligase and its use for measuring product kinetic parameters of NAD+-dependent DNA ligase from Haemophilus influenzae

Application of artificial neural network to investigate the effects of 5-fluorouracil on ribonucleotides and deoxyribonucleotides in HepG2 cells

Affinity chromatography in DNA ligase purification

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