Kinetic Analysis of Nucleotide Incorporation by Mammalian DNA Polymerase δ

Einolf, Heidi J.; Guengerich, F. Peter

doi:10.1074/jbc.m001291200

Cited by 54 publications

(91 citation statements)

References 41 publications

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“…One possibility is that PCNA that is loaded onto DNA may increase the processivity of UNG2 by decreasing the dissociation of the enzyme from DNA. Einolf and Guengerich [50] found that the K d of calf thymus DNA polymerase δ for DNA increased five-fold in the absence of PCNA. The authors concluded that PCNA appeared to affect Pol δ replication mainly by decreasing the dissociation of the polymerase from DNA [50].…”

Section: Discussionmentioning

confidence: 99%

“…Einolf and Guengerich [50] found that the K d of calf thymus DNA polymerase δ for DNA increased five-fold in the absence of PCNA. The authors concluded that PCNA appeared to affect Pol δ replication mainly by decreasing the dissociation of the polymerase from DNA [50]. A second possibility is that the UNG2·PCNA interaction serves to increase the affinity of UNG2 for (non-uracil-containing) DNA, which is relatively low.…”

Section: Discussionmentioning

confidence: 99%

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Physical and functional interaction of human nuclear uracil-DNA glycosylase with proliferating cell nuclear antigen

Bennett

2005

DNA Repair

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Uracil residues arise in DNA by the misincorporation of dUMP in place of dTMP during DNA replication or by the deamination of cytosine in DNA. Uracil-DNA glycosylase initiates DNA base excision repair of uracil residues by catalyzing the hydrolysis of the N-glycosylic bond linking the uracil base to deoxyribose. In human cells, the nuclear form of uracil-DNA glycosylase (UNG2) contains a conserved PCNA-binding motif located at the N-terminus that has been implicated experimentally in binding PCNA. Here we use purified preparations of UNG2 and PCNA to demonstrate that UNG2 physically associates with PCNA. UNG2 co-eluted with PCNA during size exclusion chromatography and bound to a PCNA affinity column. Association of UNG2 with PCNA was abolished by the addition of 100 mM NaCl, and significantly decreased in the presence of 10 mM MgCl 2 . The functional significance of the UNG2·PCNA association was demonstrated by UNG2 activity assays. Addition of PCNA (30-810 pmol) to standard uracil-DNA glycosylase reactions containing linear [uracil-3 H]DNA stimulated UNG2 catalytic activity up to 2.6-fold. UNG2 activity was also stimulated by 7.5 mM MgCl 2 . The stimulatory effect of PCNA was increased by the addition of MgCl 2 ; however, the dependence on PCNA concentration was the same, indicating that the effects of MgCl 2 and PCNA on UNG2 activity occurred by independent mechanisms. Loading of PCNA onto the DNA substrate was required for stimulation, as the activity of UNG2 on circular DNA substrates was not affected by the addition of PCNA. Addition of replication factor C and ATP to reactions containing 90 pmol of PCNA resulted in two-fold stimulation of UNG2 activity on circular DNA.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Physical and functional interaction of human nuclear uracil-DNA glycosylase with proliferating cell nuclear antigen

Bennett

2005

DNA Repair

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“…The enzyme extending SP and S 0 P 0 to form D (Table I) is assumed to be fully processive that does not dissociate after the addition of each nucleotide. Most DNA polymerase enzymes are processive where once bound, they can add more than 200 nucleotides before dissociating (Einolf and Guengerich, 2000;Lowe and Guengerich, 1996). In addition, for the short templates typical of quantitative PCR, the nucleotide incorporation error rate is expected to be negligible.…”

Section: Extensionmentioning

confidence: 99%

A kinetic model of quantitative real‐time polymerase chain reaction

Mehra

2005

Biotech & Bioengineering

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Real-time polymerase chain reaction (PCR) is one of the most sensitive and accurate methods for quantifying transcript levels especially for those expressed at low abundance. The selective amplification of target DNA over multiple cycles allows its initial concentration to be determined. The amplification rate is a complex interplay of the operating conditions, initial reactant concentrations, and reaction rate constants. Experimentally, the compounded effect of all factors is quantified in terms of an effective efficiency, which is estimated by curve fitting to the amplification data. We present a comprehensive model of PCR to study the effect of various reactant concentrations on the amplification efficiency. The model is used to calculate the kinetic progression of the target DNA concentration with cycle number under conditions when different species are stoichiometrically or kinetically limiting. The reaction efficiency remains constant for the initial cycles. As the primer concentration becomes limiting, the efficiency is marked by a gradual decrease. This is in contrast to a steep decline under nucleotide limiting conditions. Under some conditions, commonly used experimentally, increasing primer concentration has the adverse effect of reducing the final amplified template concentration. This phenomenon seen at times experimentally is explained by the simulation results under rate limiting enzyme concentrations. Primer dimer formation is shown to significantly affect the reaction rates, effective efficiency, and the estimated initial concentrations. This model, by describing the interplay of the many operating variables, will be a useful tool in designing PCR conditions and evaluating its results. ß 2005 Wiley Periodicals, Inc.

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“…However, the extent to which the mammalian forms are able to copy past chemical lesions other than photodamaged pyrimidines is largely unexplored, and the question of how lesions interact with replicative DNA polymerases is important. Recently we used purified calf thymus DNA pol ␦, considered to be the main leading strand DNA polymerase (1,2), in a series of steady-state and pre-steadystate kinetic experiments and concluded that the major features of the catalytic mechanism were very similar to those established in the prokaryotic models (21). However, questions about the processing of chemical-DNA adducts remain.…”

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confidence: 99%

Fidelity of Nucleotide Insertion at 8-Oxo-7,8-dihydroguanine by Mammalian DNA Polymerase δ

Einolf¹,

Guengerich

2001

Journal of Biological Chemistry

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130

128

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Nucleotide insertion opposite 8-oxo-7,8-dihydroguanine (8-oxoG) by fetal calf thymus DNA polymerase ␦ (pol ␦) was examined by steady-state and pre-steadystate rapid quench kinetic analyses. In steady-state reactions with the accessory protein proliferating cell nuclear antigen (PCNA), pol ␦ preferred to incorporate dCTP opposite 8-oxoG with an efficiency of incorporation an order of magnitude lower than incorporation into unmodified DNA (mainly due to an increased K m ). Pre-steady-state kinetic analysis of incorporation opposite 8-oxoG showed biphasic kinetics for incorporation of either dCTP or dATP, with rates similar to dCTP incorporation opposite G, large phosphorothioate effects (>100), and oligonucleotide dissociation apparently rate-limiting in the steady-state. Although pol ␦ preferred to incorporate dCTP (14% misincorporation of dATP) the extension past the A:8-oxoG mispair predominated. The presence of PCNA was found to be a more essential factor for nucleotide incorporation opposite 8-oxoG adducts than unmodified DNA, increased presteady-state rates of nucleotide incorporation by >2 orders of magnitude, and was essential for nucleotide extension beyond 8-oxoG. pol ␦ replication fidelity at 8-oxoG depends upon contributions from K m , K d dNTP , and rates of phosphodiester bond formation, and PCNA is an important accessory protein for incorporation and extension at 8-oxoG adducts.High fidelity DNA replication is critical to the preservation of genomic stability and the avoidance of mutations that can disrupt the regulation of complex biological systems. Cells contain several DNA polymerases and complex DNA repair systems to preserve genomic integrity (1, 2). Accurate replication is disrupted by the presence of covalent DNA-chemical adducts, which can be misread and lead to mutations and cancer (3). Understanding the miscoding events induced by modified DNA is important in understanding risks of environmental chemicals, as well as aspects of chemotherapeutic treatment. Misincorporation is primarily a kinetic phenomenon and not simply thermodynamic. Work with several DNA adducts and artificial DNA bases clearly indicates that both the identity of incorporated bases and their frequency of substitution are functions of which polymerase is used as a catalyst (4 -10). Our own work on how polymerases influence misincorporation has been focused on 8-oxoG 1 (9,(11)(12)(13)(14). 8-OxoG is a relatively simple adduct in that the only chemical attached to the DNA is one atom of oxygen, and it was selected as a model because of its relatively high mutagenicity and lack of polymerase blockage. This lesion is generally regarded as being the most abundant of those induced by oxidative damage (15-17).Polymerases derived from prokaryotic systems have been used extensively as models for mechanistic studies because of their availability, the general lack of need for complex accessory proteins, and the availability of structural and mechanistic information (18). The question arises as to how relevant findings made with the...

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Kinetic Analysis of Nucleotide Incorporation by Mammalian DNA Polymerase δ

Cited by 54 publications

References 41 publications

Physical and functional interaction of human nuclear uracil-DNA glycosylase with proliferating cell nuclear antigen

Physical and functional interaction of human nuclear uracil-DNA glycosylase with proliferating cell nuclear antigen

A kinetic model of quantitative real‐time polymerase chain reaction

Fidelity of Nucleotide Insertion at 8-Oxo-7,8-dihydroguanine by Mammalian DNA Polymerase δ

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