A false note of DNA polymerase iota in the choir of genome caretakers in mammals

Генинг, Л. В.; Makarova, Аlena V.; Malashenko, A. M.; Тарантул, В. З.

doi:10.1134/s0006297906020064

Cited by 14 publications

(23 citation statements)

References 18 publications

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“…Indeed, no pol protein was detected by Western blotting of testis extracts of 129 mice using an antibody raised against the COOH terminus of murine pol that recognizes full-length or misspliced variants of pol (26). However, a recent study (48) has reported that crude extracts of brain cells from 129͞J mice (but not extracts of other 129͞J cells) have the ability to insert dGMP opposite template T in an oligonucleotide primer template. The authors claim this misinsertion activity is due to pol rather than any of the other DNA polymerases that can insert dGMP opposite T, and they further speculate that brain cells of 129 mice contain alternatively spliced pol that retains catalytic activity.…”

Section: Discussionmentioning

confidence: 99%

Participation of mouse DNA polymerase ι in strand-biased mutagenic bypass of UV photoproducts and suppression of skin cancer

Dumstorf

Clark

Lin

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

102

101

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DNA polymerase (pol ) is a conserved Y family enzyme that is implicated in translesion DNA synthesis (TLS) but whose cellular functions remain uncertain. To test the hypothesis that pol performs TLS in cells, we compared UV-induced mutagenesis in primary fibroblasts derived from wild-type mice to mice lacking functional pol , pol , or both. A deficiency in mouse DNA polymerase (pol ) enhanced UV-induced Hprt mutant frequencies. This enhanced UV-induced mutagenesis and UV-induced mutagenesis in wild-type cells were strongly diminished in cells deficient in pol , indicating that pol participates in the bypass of UV photoproducts in cells. Moreover, a clear strand bias among UV-induced base substitutions was observed in wild-type cells that was diminished in pol -and pol -deficient mouse cells and abolished in cells deficient in both enzymes. These data suggest that these enzymes bypass UV photoproducts in an asymmetric manner. To determine whether pol status affects cancer susceptibility, we compared the UV-induced skin cancer susceptibility of wild-type mice to mice lacking functional pol , pol , or both. Although pol deficiency alone had no effect, UV-induced skin tumors in pol -deficient mice developed 4 weeks earlier in mice concomitantly deficient in pol . Collectively, these data reveal functions for pol in bypassing UV photoproducts and in delaying the onset of UV-induced skin cancer. The most compelling evidence for a role in translesion synthesis (TLS) is with pol . pol can efficiently bypass a cis-syn thymine-thymine dimer (6, 7), it localizes at replication foci after UV irradiation of mammalian cells (8), and its inactivation in XPV patients (9, 10) increases UV light-induced mutagenesis and skin cancer (reviewed in refs. 4 and 11). Increased UVinduced mutagenesis associated with pol deficiency indicates that mutagenic bypass of UV photoproducts can be catalyzed by other polymerases. One candidate is pol (2), which can bypass a thymine-thymine dimer in vitro (12) and is required for a large proportion of UV mutagenesis in vivo (2,13,14). Another candidate is pol , which physically interacts with pol and colocalizes with pol at replication foci after UV irradiation (8).Two hypotheses have been put forth regarding the outcome of putative UV photoproduct bypass by pol . One suggests that bypass should be mutagenic because of pol -catalyzed misinsertion of nucleotides opposite photoproducts (15, 16). pol is renowned for high rates of misinsertion of nucleotides opposite template pyrimidines during DNA synthesis in vitro (reviewed in ref.3). However, a study of UV-induced mutagenesis in cultured 293T cells in which pol expression was decreased by using siRNA concluded that pol has no significant role in UV lesion bypass and mutagenesis (17). A second hypothesis is that pol bypass could be antimutagenic for UV-induced C to T transition mutations. This antimutagenic hypothesis derives from a pol preference for inserting dGMP opposite template T, leading to the suggestion (18, 19) that synthesis by pol could b...

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Section: Discussionmentioning

confidence: 99%

Participation of mouse DNA polymerase ι in strand-biased mutagenic bypass of UV photoproducts and suppression of skin cancer

Dumstorf

Clark

Lin

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

102

101

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“…However, on the basis of an analytical assay designed to detect the unique mutagenic profile of Pol, Gening and colleagues (16) showed that in 129/J strain mice, Pol activity was detectable in brain extracts but not in testis. In this report, we provide extensive evidence showing that 129-derived strains are indeed not completely deficient in Pol but rather express a variant of the Pol protein that retains catalytic activity in a wide variety of tissues.…”

Section: Discussionmentioning

confidence: 99%

“…The 129-derived 129/SvJ and 129/Ola mouse strains possess a nonsense mutation in codon 27 of exon 2 in both alleles and have been reported to be naturally deficient in Pol (15). However, data from Gening and colleagues determined using a specific Pol activity assay suggested the presence of residual activity in brain extracts of 129-derived mouse strains (16).…”

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confidence: 93%

129-Derived Mouse Strains Express an Unstable but Catalytically Active DNA Polymerase Iota Variant

Aoufouchi

Smet

Delbos

et al. 2015

Molecular and Cellular Biology

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cMice derived from the 129 strain have a nonsense codon mutation in exon 2 of the polymerase iota (Pol) gene and are therefore considered Pol deficient. When we amplified Pol mRNA from 129/SvJ or 129/Ola testes, only a small fraction of the full-length cDNA contained the nonsense mutation; the major fraction corresponded to a variant Pol isoform lacking exon 2. Pol mRNA lacking exon 2 contains an open reading frame, and the corresponding protein was detected using a polyclonal antibody raised against the C terminus of the murine Pol protein. The identity of the corresponding protein was further confirmed by mass spectrometry. Although the variant protein was expressed at only 5 to 10% of the level of wild-type Pol, it retained de novo DNA synthesis activity, the capacity to form replication foci following UV irradiation, and the ability to rescue UV light sensitivity in Pol ؊/؊ embryonic fibroblasts derived from a new, fully deficient Pol knockout (KO) mouse line. Furthermore, in vivo treatment of 129-derived male mice with Velcade, a drug that inhibits proteasome function, stabilized and restored a substantial amount of the variant Pol in these animals, indicating that its turnover is controlled by the proteasome. An analysis of two xeroderma pigmentosum-variant (XPV) cases corresponding to missense mutants of Pol, a related translesion synthesis (TLS) polymerase in the same family, similarly showed a destabilization of the catalytically active mutant protein by the proteasome. Collectively, these data challenge the prevailing hypothesis that 129-derived strains of mice are completely deficient in Pol activity. The data also document, both for 129-derived mouse strains and for some XPV patients, new cases of genetic defects corresponding to the destabilization of an otherwise functional protein, the phenotype of which is reversible by proteasome inhibition.

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“…The cell extracts were prepared as previously described (Gening et al 2006;Kazakov et al 2008). The three complementary oligonucleotides (the 17 nt universal primer and two 30 mer templates) were gel purified and used to test ''misGvA'' activity.…”

Section: Assay Of Dna Polymerase Activity In Cell Extractsmentioning

confidence: 99%

“…Therefore, the search for new methods and models for analysis of expression, biochemical properties, functions, and regulation of Pol i is urgent. As a step to understanding the in vivo functions of Pol i, we recently proposed a method of determination of Pol i activity in crude cell extracts of animal tissues and human tumors which was based on the detection of Pol i-specific ''misGvA'' activity (Gening et al 2006;Kazakov et al 2008). We further increased the sensitivity of the assay by replacing Mg 2?…”

Section: Introductionmentioning

confidence: 99%

Transient expression and activity of human DNA polymerase iota in loach embryos

et al. 2011

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Human DNA polymerase iota (Pol ι) is a Y-family DNA polymerase with unusual biochemical properties and not fully understood functions. Pol ι preferentially incorporates dGTP opposite template thymine. This property can be used to monitor Pol ι activity in the presence of other DNA polymerases, e.g. in cell extracts of tissues and tumors. We have now confirmed the specificity and sensitivity of the method of Pol ι activity detection in cell extracts using an animal model of loach Misgurnus fossilis embryos transiently expressing human Pol ι. The overexpression of Pol ι was shown to be accompanied by an increase in abnormalities in development and the frequency of pycnotic nuclei in fish embryos. Further analysis of fish embryos with constitutive or regulated Pol ι expression may provide insights into Pol ι functions in vertebrate animals.

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A false note of DNA polymerase iota in the choir of genome caretakers in mammals

Cited by 14 publications

References 18 publications

Participation of mouse DNA polymerase ι in strand-biased mutagenic bypass of UV photoproducts and suppression of skin cancer

Participation of mouse DNA polymerase ι in strand-biased mutagenic bypass of UV photoproducts and suppression of skin cancer

129-Derived Mouse Strains Express an Unstable but Catalytically Active DNA Polymerase Iota Variant

Transient expression and activity of human DNA polymerase iota in loach embryos

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