Molecular Dynamics of a Food Carcinogen−DNA Adduct in a Replicative DNA Polymerase Suggest Hindered Nucleotide Incorporation and Extension

Y-family DNA polymerase iota (Pol ι) is involved in DNA damage response and tolerance. Mutations and altered expression level of POLI gene are linked to a higher incidence of cancer. We biochemically characterized five active site polymorphic variants of human Pol ι: R71G (rs3218778), P118L (rs554252419), I236M (rs3218784), E251K (rs3218783) and P365R (rs200852409). We analyzed fidelity of nucleotide incorporation on undamaged DNA, efficiency and accuracy of DNA damage bypass, as well as 5′-deoxyribophosphate lyase (dRP-lyase) activity. The I236M and P118L variants were indistinguishable from the wild-type Pol ι in activity. The E251K and P365R substitutions altered the spectrum of nucleotide incorporation opposite several undamaged DNA bases. The P365R variant also reduced the dRP-lyase activity and possessed the decreased TLS activity opposite 8-oxo-G. The R71G mutation dramatically affected the catalytic activities of Pol ι. The reduced DNA polymerase activity of the R71G variant correlated with an enhanced fidelity of nucleotide incorporation on undamaged DNA, altered lesion-bypass activity and reduced dRP-lyase activity. Therefore, this amino acid substitution likely alters Pol ι functions in vivo.

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Fidelity of Nucleotide Incorporation by Human Mitochondrial DNA Polymerase

Journal of Biological Chemistry ◽

10.1074/jbc.m106045200 ◽

2001 ◽

Vol 276 (41) ◽

pp. 38090-38096 ◽

Cited By ~ 2

Author(s):

Allison A. Johnson ◽

Kenneth A. Johnson

Keyword(s):

Mitochondrial Dna ◽

Dna Polymerase ◽

Human Mitochondrial Dna ◽

Nucleotide Incorporation ◽

Mitochondrial Dna Polymerase

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Micromethod for phosphonoformate inhibition assay of hepatitis B viral DNA polymerase.

Clinical Chemistry ◽

10.1093/clinchem/30.4.549 ◽

1984 ◽

Vol 30 (4) ◽

pp. 549-552 ◽

Cited By ~ 7

Author(s):

H J Lin ◽

P C Wu ◽

C L Lai ◽

W Chak

Keyword(s):

Hepatitis B ◽

Dna Polymerase ◽

Specific Activity ◽

Sample Volume ◽

Inhibition Assay ◽

Viral Dna ◽

Good Discrimination ◽

Human Dna ◽

Nucleotide Incorporation ◽

B Virus

Abstract A micromethod for the specific measurement of hepatitis B viral DNA polymerase in serum is presented, based on the phosphonoformate inhibition assay (J Med Virol 12: 61-70, 1983). In the micromethod, sample volume is reduced to 120 microL and the ultracentrifugation step is eliminated. The method allows good discrimination between serum infected with hepatitis B virus and uninfected serum. The cutoff value for rate of nucleotide incorporation, based on assays of 41 serum specimens negative for hepatitis B serological markers, was about 15 nU/L (90th percentile). Serum containing hepatitis B surface and antigens exhibited rates of phosphonoformate-inhibitive nucleotide incorporation of 150 (SD 150) nU/L, with an upper 90th percentile range of 17 to 667 nU/L (n = 41). The micromethod makes use of commercially available [32P]dCTP (specific activity about 7000 kCi/mol). 125I-labeled dCTP was found to be unsuitable for this assay. Human DNA polymerases in serum are detected by this method but are excluded from the phosphonoformate-inhibitive fraction.

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