Steady‐State Kinetic Analysis of DNA Polymerase Single‐Nucleotide Incorporation Products

O’Flaherty, Derek K.; Guengerich, F. Peter

doi:10.1002/0471142700.nc0721s59

Cited by 25 publications

(25 citation statements)

References 23 publications

(28 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Steady-state Kinetics-Steady-state kinetic assays were performed as described previously (23)(24)(25). The oligonucleotides used in this study are listed in Table 1.…”

Section: Methodsmentioning

confidence: 99%

Structural and Kinetic Analysis of Nucleoside Triphosphate Incorporation Opposite an Abasic Site by Human Translesion DNA Polymerase η

Patra¹,

Zhang²,

Lei

et al. 2015

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Background: Abasic sites are the most common lesion in DNA. Results: Kinetic and mass spectrometric assays demonstrate that human polymerase (pol) preferentially inserts A and G opposite an abasic site. Conclusion: Crystal structures reveal H-bonding between incoming ATP and GTP and the 5Ј-phosphate of the abasic moiety. Significance: Abasic site bypass by pol follows a "purine rule" for insertion, with formation of frameshifts.

show abstract

“…Steady-state Kinetics-Steady-state kinetic assays were performed as described previously (23)(24)(25). The oligonucleotides used in this study are listed in Table 1.…”

Section: Methodsmentioning

confidence: 99%

Structural and Kinetic Analysis of Nucleoside Triphosphate Incorporation Opposite an Abasic Site by Human Translesion DNA Polymerase η

Patra¹,

Zhang²,

Lei

et al. 2015

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

show abstract

“…Reactions were conducted for the indicated times and stopped by addition of a quench solution (95% formamide (v/v) and 10 mM EDTA). The products were loaded onto 18% (w/v) denaturing polyacrylamide gels, separated, and visualized using a Typhoon system (GE Healthcare) (63,64).…”

Section: Extension Assaysmentioning

confidence: 99%

“…For the pre-steady-state kinetic assays, 2 M dTdT: rArA or CPD:rArA complex and 437 nM RNase H2 (both in incision buffer) were mixed in a KinTek RP-3 instrument (KinTek Corporation, Austin, TX), allowing the reaction to proceed for only a very short period time. The products were separated on 18% (w/v) denaturing polyacrylamide gels and visualized with a Typhoon system (GE Healthcare) (63,64). The program ImageJ was used for quantitation, and the pre-steadystate kinetic assay data were fit to a burst equation: y ϭ A(1e Ϫkpt ) ϩ k ss E 0 t, using GraphPad Prism software (GraphPad, San Diego, CA), where A represents the apparent concentration of the active form of the enzyme, k p is the burst rate, k ss is the steady-state rate, t is time, and E 0 is the total enzyme concentration (63,64).…”

Section: Activities Of Human Dna Polymerasementioning

confidence: 99%

See 1 more Smart Citation

Human DNA polymerase η has reverse transcriptase activity in cellular environments

Ghodke

Egli

et al. 2019

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Edited by Patrick SungClassical DNA and RNA polymerase (pol) enzymes have defined roles with their respective substrates, but several pols have been found to have multiple functions. We reported previously that purified human DNA pol (hpol ) can incorporate both deoxyribonucleoside triphosphates (dNTPs) and ribonucleoside triphosphates (rNTPs) and can use both DNA and RNA as substrates. X-ray crystal structures revealed that two pol residues, Phe-18 and Tyr-92, behave as steric gates to influence sugar selectivity. However, the physiological relevance of these phenomena has not been established. Here, we show that purified hpol adds rNTPs to DNA primers at physiological rNTP concentrations and in the presence of competing dNTPs. When two rATPs were inserted opposite a cyclobutane pyrimidine dimer, the substrate was less efficiently cleaved by human RNase H2. Human XP-V fibroblast extracts, devoid of hpol , could not add rNTPs to a DNA primer, but the expression of transfected hpol in the cells restored this ability. XP-V cell extracts did not add dNTPs to DNA primers hybridized to RNA, but could when hpol was expressed in the cells. HEK293T cell extracts could add dNTPs to DNA primers hybridized to RNA, but lost this ability if hpol was deleted. Interestingly, a similar phenomenon was not observed when other translesion synthesis (TLS) DNA polymerases-hpol , , or -were individually deleted. These results suggest that hpol is one of the major reverse transcriptases involved in physiological processes in human cells.

show abstract

“…Steady-state kinetic experiments were conducted as previously described. 37–40 Briefly, assays were generally performed at 37 °C in 40 mM Tris-HCl buffer (pH 7.5) containing 100 mM KCl, 5% glycerol (v/v), 10 mM dithiothreitol (DTT), 5 mM MgCl 2 and 100 μg mL −1 bovine serum albumin (BSA). The 5′-labelled 6-carboxyfluorescein (FAM) primer-template (9-/13-mer) duplex (5 μM) was extended using 1.9 to 500 nM concentrations of hPol η in the presence of various concentrations of a single dNTP (0 to 1 mM, at 7–10 different dNTP concentrations) at 37 °C for 5–20 min.…”

Section: Methodsmentioning

confidence: 99%

Backbone Flexibility Influences Nucleotide Incorporation by Human Translesion DNA Polymerase η opposite Intrastrand Cross-Linked DNA

et al. 2015

Self Cite

View full text Add to dashboard Cite

Intrastrand cross-links (IaCL) connecting two purine nucleobases in DNA pose a challenge to high fidelity replication in the cell. Various repair pathways or polymerase bypass can cope with these lesions. The influence of the phosphodiester linkage between two neighbouring 2′-deoxyguanosine (dG) residues attached through the O6-atoms by an alkylene linker on bypass with human DNA polymerase η (hPol η) was explored in vitro. Steady-state kinetics and mass spectrometry analysis of products from nucleotide incorporation revealed that although hPol η is capable of bypassing the 3′-dG in a mostly error-free fashion, significant misinsertion was observed for the 5′-dG of the IaCL containing a butylene or heptylene linker. The lack of the phosphodiester linkage triggered a significant increase in frameshift adduct formation across the 5′-dG by hPol η, in comparison to the 5′-dG of IaCL DNA containing the phosphodiester group.

show abstract

Steady‐State Kinetic Analysis of DNA Polymerase Single‐Nucleotide Incorporation Products

Cited by 25 publications

References 23 publications

Structural and Kinetic Analysis of Nucleoside Triphosphate Incorporation Opposite an Abasic Site by Human Translesion DNA Polymerase η

Structural and Kinetic Analysis of Nucleoside Triphosphate Incorporation Opposite an Abasic Site by Human Translesion DNA Polymerase η

Human DNA polymerase η has reverse transcriptase activity in cellular environments

Backbone Flexibility Influences Nucleotide Incorporation by Human Translesion DNA Polymerase η opposite Intrastrand Cross-Linked DNA

Contact Info

Product

Resources

About