Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.7 (DNA polymerase)
17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The 1,N6-ethenodeoxyadenosine (epsilon dA) lesion is promutagenic and has been implicated in carcinogenesis. We show here that human Pol iota, a Y-family DNA polymerase, can promote replication through this lesion by proficiently incorporating a nucleotide opposite it. The structural basis of this action is rotation of the epsilon dA adduct to the syn conformation in the Pol iota active site and presentation of its 'Hoogsteen edge' for hydrogen-bonding with incoming dTTP or dCTP. We also show that Pol zeta carries out the subsequent extension reaction and that efficiency of extension from epsilon dA x T is notably higher than from epsilon dA x C. Together, our studies reveal for the first time how the exocyclic epsilon dA adduct is accommodated in a DNA polymerase active site, and they show that the combined action of Pol iota and Pol zeta provides for efficient and error-free synthesis through this potentially carcinogenic DNA lesion.
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PMID:Hoogsteen base pair formation promotes synthesis opposite the 1,N6-ethenodeoxyadenosine lesion by human DNA polymerase iota. 1681 16

Heterocyclic arylamines are highly mutagenic and cause tumors in animal models. The mutagenicity is attributed to the C8- and N2-G adducts, the latter of which accumulates due to slower repair. The C8- and N 2-G adducts derived from 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) were placed at the G1 and G3 sites of the NarI sequence, in which the G3 site is an established hot spot for frameshift mutation with the model arylamine derivative 2-acetylaminofluorene but G1 is not. Human DNA polymerase (pol) eta extended primers beyond template G-IQ adducts better than did pol kappa and much better than pol iota or delta. In 1-base incorporation studies, pol eta inserted C and A, pol iota inserted T, and pol kappa inserted G. Steady-state kinetic parameters were measured for these dNTPs opposite the C8- and N 2-IQ adducts at both sites, being most favorable for pol eta. Mass spectrometry of pol eta extension products revealed a single major product in each of four cases; with the G1 and G3 C8-IQ adducts, incorporation was largely error-free. With the G3 N 2-IQ adduct, a -2 deletion occurred at the site of the adduct. With the G1 N 2-IQ adduct, the product was error-free at the site opposite the base and then stalled. Thus, the pol eta products yielded frame-shifts with the N 2 but not the C8 IQ adducts. We show a role for pol eta and the complexity of different chemical adducts of IQ, DNA position, and DNA polymerases.
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PMID:Biochemical basis of genotoxicity of heterocyclic arylamine food mutagens: Human DNA polymerase eta selectively produces a two-base deletion in copying the N2-guanyl adduct of 2-amino-3-methylimidazo[4,5-f]quinoline but not the C8 adduct at the NarI G3 site. 1683 18

Xeroderma pigmentosum variant (XPV) patients have normal DNA excision repair, yet are predisposed to develop sunlight-induced cancer. They exhibit a 25-fold higher than normal frequency of UV-induced mutations and very unusual kinds (spectrum), mainly transversions. The primary defect in XPV cells is the lack of functional DNA polymerase (Pol) eta, the translesion synthesis DNA polymerase that readily inserts adenine nucleotides opposite photoproducts involving thymine. The high frequency and striking difference in kinds of UV-induced mutations in XPV cells strongly suggest that, in the absence of Pol eta, an abnormally error-prone polymerase substitutes. In vitro replication studies of Pol iota show that it replicates past 5'T-T3' and 5'T-U3' cyclobutane pyrimidine dimers, incorporating G or T nucleotides opposite the 3' nucleotide. To test the hypothesis that Pol iota causes the high frequency and abnormal spectrum of UV-induced mutations in XPV cells, we identified an unlimited lifespan XPV cell line expressing two forms of Pol iota, whose frequency of UV-induced mutations is twice that of XPV cells expressing one form. We eliminated expression of one form and compared the parental cells and derivatives for the frequency and kinds of UV-induced mutations. All exhibited similar sensitivity to the cytotoxicity of UV((254 nm)), and the kinds of mutations induced were identical, but the frequency of mutations induced in the derivatives was reduced to </=50% that of the parent. These data strongly support the hypothesis that in cells lacking Pol eta, Pol iota is responsible for the high frequency and abnormal spectrum of UV-induced mutations, and ultimately their malignant transformation.
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PMID:Evidence that in xeroderma pigmentosum variant cells, which lack DNA polymerase eta, DNA polymerase iota causes the very high frequency and unique spectrum of UV-induced mutations. 1740 8

Endogenously generated reactive oxygen species and genotoxic carcinogens can covalently modify bases in cellular DNA. If not recognized and removed prior to S-phase of the cell cycle, such modifications can block DNA replication fork progression. If blocked forks are not are not resolved, they result in double strand breaks and cell death. Recent data indicate that the process of translesion DNA synthesis (TLS) is a highly conserved mechanism for bypassing lesions in template DNA. Although not fully understood, in yeast a ubiquitin ligase (RAD18) signals error-prone Y family polymerases to the blocked fork to bypass the damage with potentially mutagenic consequences. Homologs of the yeast proteins are found in higher eukaryotic cells, including human. We are examining the hypothesis that RAD18 acts as a proximal signal to Y-family polymerases to bypass damage, in a manner analogous to yeast but with additional layers of complexity. Here we report that RAD18 accumulates in nuclear foci after UV irradiation only in cells entering S-phase with DNA damage. These foci co-localize with proliferating cell nuclear antigen (PCNA). In addition, a newly described DNA polymerase, pol iota, also forms nuclear foci in a damage- and S-phase dependent manner. These data support our overall hypothesis that RAD18 accumulates at blocked forks and initiates the signal to recruit TLS polymerases.
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PMID:RAD18 signals DNA polymerase IOTA to stalled replication forks in cells entering S-phase with DNA damage. 1829 Mar 23

We analyzed the translesion synthesis across the UV-induced lesions, the (6-4) photoproduct and its Dewar valence isomer, by using human DNA polymerases eta and iota in vitro. The primer extension experiments revealed that pol eta tended to incorporate dG opposite the 3' component of both lesions, but the incorporation efficiency for the Dewar isomer was higher than that for the (6-4) photoproduct. On the other hand, pol iota was likely to incorporate dA opposite the 3' components of the (6-4) photoproduct and its Dewar isomer with a similar efficiency. Elongation after the incorporation opposite the UV lesions was not observed for these Y-family polymerases. We further analyzed the bypass ability of an engineered polymerase developed from Thermus DNA polymerase for the amplification of ancient DNA. This polymerase could bypass the Dewar isomer more efficiently than the (6-4) photoproduct.
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PMID:Translesion synthesis across the (6-4) photoproduct and its Dewar valence isomer by the Y-family and engineered DNA polymerases. 1877 92

DNA polymerase (pol) iota, a member of the mammalian Y-family of DNA polymerases involved in translesion DNA synthesis, has been previously suggested to peculiarly utilize Hoogsteen base pairing for DNA synthesis opposite template purines, unlike pols eta and kappa, which utilize Watson-Crick (W-C) base pairing. To investigate the possible roles of Hoogsteen, W-C, and wobble base-pairing modes in the selection of nucleotides opposite template pyrimidines by human pol iota, we carried out kinetic analyses of incorporation of modified purine nucleoside triphosphates including 7-deazapurines, inosine, 2-aminopurine, 2,6-diaminopurine, and 6-chloropurine, which affect H-bonding in base-pair formation opposite template pyrimidines. Carbon substitution at the N7 atom of purine nucleoside triphosphates, which disrupts Hoogsteen base pairing, only slightly inhibited DNA synthesis opposite template pyrimidines by pol iota, which was not substantially different from human pols eta and kappa. Opposite template T, only the relative wobble stabilities (inferred from the potential numbers of H-bonding, steric, and electrostatic interactions but not measured) of base pairs were positively correlated to the relative efficiencies of nucleotide incorporation by pol iota but not the relative W-C or Hoogsteen stabilities, unlike pols eta and kappa. In contrast, opposite C, only the relative W-C stabilities of base pairs were positively correlated to the relative efficiencies of nucleotide incorporation by pol iota, as with pols eta and kappa. These results suggest that pol iota might not indispensably require Hoogsteen base pairing for DNA synthesis opposite pyrimidines but rather might prefer wobble base pairing in the selection of nucleotides opposite T and W-C base pairing opposite C.
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PMID:Kinetic analysis of base-pairing preference for nucleotide incorporation opposite template pyrimidines by human DNA polymerase iota. 1937 29

An increased level of mutagenesis, partially caused by imbalanced activities of error prone DNA polymerases, is a key symptom of cell malignancy. To clarify the possible role of incorrect DNA polymerase iota (Pol iota) function in increased frequency of mutations in mammalian cells, the activity of this enzyme in extracts of cells of different mouse organs and human eye (melanoma) and eyelid (basal-cell skin carcinoma) tumor cells was studied. Both Mg2+, considered as the main activator of the enzyme reaction of in vivo DNA replication, and Mn2+, that activates homogeneous Pol iota preparations in experiments in vitro more efficiently compared to all other bivalent cations, were used as cofactors of the DNA polymerase reaction in these experiments. In the presence of Mg2+, the enzyme was active only in cell extracts of mouse testicles and brain, whereas in the presence of Mn2+ the activity of Pol iota was found in all studied normal mouse organs. It was found that in cell extracts of both types of malignant tumors (basal-cell carcinoma and melanoma) Pol iota activity was observed in the presence of either Mn2+ or Mg2+. Manganese ions activated Pol iota in both cases, though to a different extent. In the presence of Mn2+ the Pol iota activity in the basal-cell carcinoma exceeded 2.5-fold that in control cells (benign tumors from the same eyelid region). In extracts of melanoma cells in the presence of either cation, the level of the enzyme activity was approximately equal to that in extracts of cells of surrounding tumor-free tissues as well as in eyes removed after traumas. The distinctive feature of tissue malignancy (in basal-cell carcinoma and in melanoma) was the change in DNA synthesis revealed as Mn2+-activated continuation of DNA synthesis after incorrect incorporation of dG opposite dT in the template by Pol iota. Among cell extracts of different normal mouse organs, only those of testicles exhibited a similar feature. This similarity can be explained by cell division blocking that occurs in all normal cells except in testicles and in malignant cells.
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PMID:Effect of human cell malignancy on activity of DNA polymerase iota. 2067 15


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