L. V. Gening

Inaccurate DNA Synthesis in Cell Extracts of Yeast Producing Active Human DNA Polymerase Iota

Mammalian Pol ι has an unusual combination of properties: it is stimulated by Mn2+ ions, can bypass some DNA lesions and misincorporates “G” opposite template “T” more frequently than incorporates the correct “A.” We recently proposed a method of detection of Pol ι activity in animal cell extracts, based on primer extension opposite the template T with a high concentration of only two nucleotides, dGTP and dATP (incorporation of “G” versus “A” method of Gening, abbreviated as “misGvA”). We provide unambiguous proof of the “misGvA” approach concept and extend the applicability of the method for the studies of variants of Pol ι in the yeast model system with different cation cofactors. We produced human Pol ι in bakers yeast, which do not have a POLI ortholog. The “misGvA” activity is absent in cell extracts containing an empty vector, or producing catalytically dead Pol ι, or Pol ι lacking exon 2, but is robust in the strain producing wild-type Pol ι or its catalytic core, or protein with the active center L62I mutant. The signature pattern of primer extension products resulting from inaccurate DNA synthesis by extracts of cells producing either Pol ι or human Pol η is different. The DNA sequence of the template is critical for the detection of the infidelity of DNA synthesis attributed to DNA Pol ι. The primer/template and composition of the exogenous DNA precursor pool can be adapted to monitor replication fidelity in cell extracts expressing various error-prone Pols or mutator variants of accurate Pols. Finally, we demonstrate that the mutation rates in yeast strains producing human DNA Pols ι and η are not elevated over the control strain, despite highly inaccurate DNA synthesis by their extracts.

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

DNA polymerase iota (Polι) of mammals is a member of the Y family of DNA polymerases. Among many other genome caretakers, these enzymes are responsible for maintaining genome stability. The members of the Y-family DNA polymerases take part in translesion DNA synthesis, bypassing some DNA lesions, and are characterized by low fidelity of DNA synthesis. A unique ability of Polι to predominantly incorporate G opposite T allowed us to identify the product of this enzyme among those synthesized by other DNA polymerases. This product can be called a “false note” of Polι. We measured the enzyme activity of Polι in crude extracts of cells from different organs of five inbred strains of mice (C3H/Sn, 101/H, C57BL/6, BALB/c, 129/J) that differed in a number of parameters. The “false note” of Polι was clearly sounding only in the extracts of testis and brain cells from four analyzed strains: C3H/Sn, 101/H, C57BL/6, BALB/c. In mice of 129/J strain that had a nonsense mutation in the second exon of the polι gene, the Polι activity was reliably detectable only in the extracts of brain. The data show that the active enzyme can be formed in some cell types even if they carry a nonsense mutation in the polι gene. This supports tissue-specific regulation of polι gene expression through alternative splicing. A semiquantitative determination of Polι activity in mice strains different in their radiosensitivity suggests a reciprocal correlation between the enzyme activity of Polι in testis and the resistance of mice to radiation.

Effect of human cell malignancy on activity of DNA polymerase ι

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 ι (Pol ι) 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 ι 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 ι 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 ι activity was observed in the presence of either Mn2+ or Mg2+. Manganese ions activated Pol ι in both cases, though to a different extent. In the presence of Mn2+ the Pol ι 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 ι. 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.

DNA polymerase iota-like activity in crude cell extracts of different mouse organs.

The recently discovered DNA polymerase iota differs greatly from the numerous eukaryotic and prokaryotic DNA polymerases known previously in its ability to catalyze error-prone DNA synthesis. Using homogeneous preparations of the enzyme, it was shown previously that DNA polymerase iota incorporated preferentially dGMP opposite the thymidine of the template in the growing DNA chain. To elucidate the role of this enzyme in the mammals, its activity was assayed in crude cell extracts of different mouse organs. It is shown that the extracts of the brain and testis cells exhibit the highest activity of DNA polymerase iota, which is not in agreement with the results of other authors. The data suggest that the tissue specific expression of DNA polymerase iota is regulated to a significant degree at the posttranscriptional and posttranslational levels.

Isolation and Characterization of High Affinity Aptamers Against DNA Polymerase Iota

Human DNA-polymerase iota (Pol ι) is an extremely error-prone enzyme and the fidelity depends on the sequence context of the template. Using the in vitro systematic evolution of ligands by exponential enrichment (SELEX) procedure, we obtained an oligoribonucleotide with a high affinity to human Pol ι, named aptamer IKL5. We determined its dissociation constant with homogenous preparation of Pol ι and predicted its putative secondary structure. The aptamer IKL5 specifically inhibits DNA-polymerase activity of the purified enzyme Pol ι, but did not inhibit the DNA-polymerase activities of human DNA polymerases beta and kappa. IKL5 suppressed the error-prone DNA-polymerase activity of Pol ι also in cellular extracts of the tumor cell line SKOV-3. The aptamer IKL5 is useful for studies of the biological role of Pol ι and as a potential drug to suppress the increase of the activity of this enzyme in malignant cells.

Evolution of DNA polymerase ι structure and function in eukaryotes

Analysis of DNA polymerase ι (Pol ι) enzymic activity in different classes of eukaryotes has shown that error-prone activity of this enzyme can be found only in mammals, and that it is completely absent from organisms that are at lower stages of development. It was supposed that the emergence of the error-prone Pol ι activity in mammals is caused by structural alteration of the active center. Possible functions of error-prone Pol ι in higher eukaryotes are discussed.

Association between high activity of DNA polymerase iota and the development of human uveal melanoma

Enzymatic activity of DNA polymerase iota (Pol ι) was analyzed in human uveal melanoma cell extracts, using an earlier elaborated approach. The Pol ι activity was observed in seven out of eight malignant tumors, while it was absent in the normal uveal tract cells of the same patients. These findings serve as an additional confirmation of the Pol ι oncogenic potential.

Alternative splicing at exon 2 results in the loss of the catalytic activity of mouse DNA polymerase iota in vitro

Y-family DNA polymerase iota (Pol ι) possesses both DNA polymerase and dRP lyase activities and was suggested to be involved in DNA translesion synthesis and base excision repair in mammals. The 129 strain of mice and its derivatives have a natural nonsense codon mutation in the second exon of the Pol ι gene resulting in truncation of the Pol ι protein. These mice were widely used as a Pol ι-null model for in vivo studies of the Pol ι function. However whether 129-derived strains of mice are fully deficient in the Pol ι functions was a subject of discussion since Pol ι mRNA undergoes alternative splicing at exon 2. Here we report purification of mouse Pol ι lacking the region encoded by exon 2, which includes several conserved residues involved in catalysis. We show that the deletion abrogates both the DNA polymerase and dRP lyase activities of Pol ι in the presence of either Mg2+ or Mn2+ ions. Thus, 129-derived strains of mice express catalytically inactive alternatively spliced Pol ι variant, whose cellular functions, if any exist, remain to be established.

Effect of Mn(II) on error-prone DNA polymerase iota activity in extracts from human normal and tumor cells

DNA polymerase iota (Pol ι) which has some peculiar features and is characterized by an extremely error prone DNA synthesis, belongs to the group of enzymes preferentially activated by Mn2+ instead of Mg2+. In this work, using misGvA method (misincorporation of “G” versus “A”, method of Gening) we studied the effect of Mn2+ on DNA synthesis in cell extracts from a—normal human and murine tissues, b—human tumor (uveal melanoma), and c—cultured human tumor cell lines SKOV-3 and HL-60. Each group displayed characteristic features of Mn-dependent DNA synthesis. Changes in Mn-dependent DNA synthesis caused by malignant transformation of normal tissues are described. It was also shown that the error prone DNA synthesis catalyzed by Pol ι in extracts of all cell types was efficiently suppressed by an RNA aptamer (IKL5) against Pol ι obtained by us earlier. The results obtained suggest that IKL5 might be used to suppress enhanced activity of Pol ι in tumor cells.

DNA polymerase ι of mammals as a participant in translesion synthesis of DNA

This review describes the properties of some specialized DNA polymerases participating in translesion synthesis of DNA. Special attention is given to these properties in vivo. DNA polymerase iota (Polι) of mammals has very unusual features and is extremely error-prone. Based on available data, a hypothesis is proposed explaining how mammalian cells can explore the unusual features of DNA Polι to bypass DNA damages and to simultaneously prevent its mutagenic potential.