Human DNA Polymerase ν Catalyzes Correct and Incorrect DNA Synthesis with High Catalytic Efficiency

Gowda, A. S. Prakasha; Moldovan, George‐Lucian; Spratt, Thomas E.

doi:10.1074/jbc.m115.653287

Cited by 8 publications

(28 citation statements)

References 59 publications

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“…These values are similar to a k pol of 100 s −1 and a K d dNTP of 20 μ M found previously. 35 Misincorporation occurs with k pol values of 10 to 32 s −1 and apparent K d dNTP values of 47 to 136 μ M. As we previously observed, the amplitude of the rapid reaction was also dependent on dNTP concentrations. The A max /[pol] values were similar for all dNTP/template pairings, and ranged from 0.5 to 0.74.…”

Section: Resultssupporting

confidence: 53%

“…35 For example, the A-family T7 DNA polymerase forms are inactive during the insertion of dCTP and dTTP opposite O 6 -Me-dG. 38 The decreased burst amplitudes associated with the pol ν bypass of the four adducts studied in this article can potentially be due to this phenomena.…”

Section: Resultsmentioning

confidence: 88%

“…At this concentration of DNA and polymerase, 95% of the DNA should be bound to polymerase, assuming that the modification does not alter the K d of the pol to undamaged DNA. 35 dCTP is incorporated opposite dG with a half-life of 40 ms, while the incorporation opposite O 6 -Me-dG is only slightly slower with a half-life of 60 ms. The incorporation of dTTP opposite O 6 -Me-dG is considerably slower with a half-life of 1 s. The incorporation of dCTP opposite O 6 -POB-dG is extremely slow with a half-life of >1000 s. With dT modification, both O 2 -Me-dT and O 2 -POB-dT reacted very slowly.…”

Section: Resultsmentioning

confidence: 99%

“…35 In addition, pol ν produces G/T mispairs at a high frequency, in a sequence-specific manner. 27,39 The current paradigm is that high fidelity polymerases use geometric selection to favor the formation of the Watson–Crick base pairs.…”

Section: Discussionmentioning

confidence: 99%

“…The primer was 32 P-labeled with [ γ - 32 P]ATP and annealed with a 20% excess of the template as previously described. 35 …”

Section: Methodsmentioning

confidence: 99%

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DNA Polymerase ν Rapidly Bypasses O⁶-Methyl-dG but Not O⁶-[4-(3-Pyridyl)-4-oxobutyl-dG and O²-Alkyl-dTs

Gowda

Spratt

2016

Chem. Res. Toxicol.

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4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent tobacco carcinogen that forms mutagenic DNA adducts including O6-methyl-2′-deoxyguanosine (O6-Me-dG), O6-[4-(3-pyridyl)-4-oxobut-1-yl]-dG (O6-POB-dG), O2-methylthymidine (O2-Me-dT), and O2-POB-dT. We evaluated the ability of human DNA polymerase ν to bypass this damage to evaluate the structural constraints on substrates for pol ν and to evaluate if there is kinetic evidence suggesting the in vivo activity of pol ν on tobacco-induced DNA damage. Presteady-state kinetic analysis has indicated that O6-Me-dG is a good substrate for pol ν, while O6-POB-dG and the O2-alkyl-dT adducts are poor substrates for pol ν. The reactivity with O6-Me-dG is high with a preference for dCTP > dGTP > dATP > dTTP. The catalytic activity of pol ν toward O6-Me-dG is high and can potentially be involved in its bypass in vivo. In contrast, pol ν is unlikely to bypass O6-POB-dG or the O2-alkyl-dTs in vivo.

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Section: Resultssupporting

confidence: 53%

Section: Resultsmentioning

confidence: 88%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

“…The primer was 32 P-labeled with [ γ - 32 P]ATP and annealed with a 20% excess of the template as previously described. 35 …”

Section: Methodsmentioning

confidence: 99%

See 3 more Smart Citations

DNA Polymerase ν Rapidly Bypasses O⁶-Methyl-dG but Not O⁶-[4-(3-Pyridyl)-4-oxobutyl-dG and O²-Alkyl-dTs

Gowda

Spratt

2016

Chem. Res. Toxicol.

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Divalent Cations Alter the Rate-Limiting Step of PrimPol-Catalyzed DNA Elongation

Zhao

Morehouse

et al. 2019

Journal of Molecular Biology

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PrimPol is the most recently discovered human DNA polymerase/primase and plays an emerging role in nuclear and mitochondrial genomic maintenance. As a member of archaeo-eukaryotic primase (AEP) superfamily enzymes, PrimPol possesses DNA polymerase and primase activities that are important for replication fork progression in vitro and in cellulo. The enzymatic activities of PrimPol are critically dependent on the nucleotidyl-transfer reaction to incorporate deoxyribonucleotides successively; however, our knowledge concerning the kinetic mechanism of the reaction remains incomplete. Using enzyme kinetic analyses and computer simulations, we dissected the mechanism by which PrimPol transfers a nucleotide to a primer-template DNA, which comprises DNA binding, conformational transition, nucleotide binding, phosphoester bond formation, and dissociation steps. We obtained the rate constants of the steps by steady-state and pre-steady-state kinetic analyses and simulations. Our data demonstrate that the rate-limiting step of PrimPol-catalyzed DNA elongation depends on the metal cofactor involved. In the presence of Mn 2+ , a conformational transition step from non-productive to productive PrimPol:DNA complexes limits the enzymatic turnover, whereas, in the presence of Mg 2+ , the chemical step becomes rate-limiting. As evidenced from our kinetic and simulation data, PrimPol maintains the same kinetic mechanism under either millimolar or physiological micromolar Mn 2+ concentration. Our study revealed the underlying mechanism by which PrimPol catalyzes nucleotide incorporation with two common metal cofactors, and provides a kinetic basis for further understanding the regulatory mechanism of this functionally diverse primase-polymerase.

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DNA Polymerases η and ζ Combine to Bypass O²-[4-(3-Pyridyl)-4-oxobutyl]thymine, a DNA Adduct Formed from Tobacco Carcinogens

Gowda

Spratt

2016

Chem. Res. Toxicol.

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4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N′-nitrosonornicotine (NNN) are important human carcinogens in tobacco products. They are metabolized to produce a variety 4-(3-pyridyl)-4-oxobutyl (POB) DNA adducts including O2-[4-(3-pyridyl)-4-oxobut-1-yl]thymidine (O2-POB-dT), the most abundant POB adduct in NNK- and NNN-treated rodents. To evaluate the mutagenic properties of O2-POB-dT, we measured the rate of insertion of dNTPs opposite and extension past O2-POB-dT and O2-Me-dT by purified human DNA polymerases η, κ, ι, and yeast polymerase ζ in vitro. Under conditions of polymerase in excess, polymerase η was most effective at the insertion of dNTPs opposite O2-alkyl-dTs. The time courses were biphasic suggesting the formation of inactive DNA-polymerase complexes. The kpol parameter was reduced approximately 100-fold in the presence of the adduct for pol η, κ, and ι. Pol η was the most reactive polymerase for the adducts due to a higher burst amplitude. For all three polymerases, the nucleotide preference was dATP > dTTP ≫ dGTP and dCTP. Yeast pol ζ was most effective in bypassing the adducts; the kcat/Km values were reduced only 3-fold in the presence of the adducts. The identity of the nucleotide opposite the O2-alkyl-dT did not significantly affect the ability of pol ζ to bypass the adducts. The data support a model in which pol η inserts ATP or dTTP opposite O2-POB-dT, and then, pol ζ extends past the adduct.

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Human DNA Polymerase ν Catalyzes Correct and Incorrect DNA Synthesis with High Catalytic Efficiency

Cited by 8 publications

References 59 publications

DNA Polymerase ν Rapidly Bypasses O⁶-Methyl-dG but Not O⁶-[4-(3-Pyridyl)-4-oxobutyl-dG and O²-Alkyl-dTs

DNA Polymerase ν Rapidly Bypasses O⁶-Methyl-dG but Not O⁶-[4-(3-Pyridyl)-4-oxobutyl-dG and O²-Alkyl-dTs

Divalent Cations Alter the Rate-Limiting Step of PrimPol-Catalyzed DNA Elongation

DNA Polymerases η and ζ Combine to Bypass O²-[4-(3-Pyridyl)-4-oxobutyl]thymine, a DNA Adduct Formed from Tobacco Carcinogens

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Human DNA Polymerase ν Catalyzes Correct and Incorrect DNA Synthesis with High Catalytic Efficiency

Cited by 8 publications

References 59 publications

DNA Polymerase ν Rapidly Bypasses O6-Methyl-dG but Not O6-[4-(3-Pyridyl)-4-oxobutyl-dG and O2-Alkyl-dTs

DNA Polymerase ν Rapidly Bypasses O6-Methyl-dG but Not O6-[4-(3-Pyridyl)-4-oxobutyl-dG and O2-Alkyl-dTs

Divalent Cations Alter the Rate-Limiting Step of PrimPol-Catalyzed DNA Elongation

DNA Polymerases η and ζ Combine to Bypass O2-[4-(3-Pyridyl)-4-oxobutyl]thymine, a DNA Adduct Formed from Tobacco Carcinogens

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Product

Resources

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DNA Polymerase ν Rapidly Bypasses O⁶-Methyl-dG but Not O⁶-[4-(3-Pyridyl)-4-oxobutyl-dG and O²-Alkyl-dTs

DNA Polymerase ν Rapidly Bypasses O⁶-Methyl-dG but Not O⁶-[4-(3-Pyridyl)-4-oxobutyl-dG and O²-Alkyl-dTs

DNA Polymerases η and ζ Combine to Bypass O²-[4-(3-Pyridyl)-4-oxobutyl]thymine, a DNA Adduct Formed from Tobacco Carcinogens