Alterations in Synthesis and Repair of DNA during the Development of Loach Misgurnus fossilis

Abstract

Using a modified radiolabeled primer extension method (we named this modification misGvA-"misincorporation of G versus A") we have investigated the DNA synthesis and repair at early and late stages of development of loach Misgurnus fossilis. The misincorporation activity of DNA polymerase iota (Pol ι) in wild-type loach could not be detected by this method at any stage of loach development. In transgenic loach overexpressing human Pol ι we have shown that the bypassing of DNA synthesis arrest after incorporation of mismatched nucleotide by Pol ι (the T-stop) was not associated with this enzyme. Non-transgenic loach larvae are virtually lacking the capacity for error correction of DNA duplex containing a mismatched nucleotide. Such repair activity develops only in the adult fish. It appears that the initial stages of development are characterized by more intensive DNA synthesis, while in terminal stages the repair activities become more prominent. The misGvA approach clearly indicates substantial changes in the DNA synthesis intensity, although the role of particular replicative and repair DNA polymerases in this process requires further study.

Keywords: DNA polymerase iota; DNA repair; DNA synthesis; loach.

Figure 1

DNA synthesis in extracts of transgenic loach larvae expressing human Pol ι. In all cases, quantitative parameters were determined from the results of three to five independent experiments. (A) Alternative DNA synthesis products, generated in vertebrate cell extracts with substrate No. 1. The frame highlights the nucleotides that are incorporated into substrate No. 1 during DNA synthesis; (B) Electropherogram of DNA synthesis products using substrate No. 1, generated in cell extracts of transgenic loach larvae, that either express inactive (lane 1) or active form of Pol ι (lane 2). The products incorporating mismatched G that results from Pol ι activity correspond to bands that migrate more slowly in gel; (C) Histogram representing total main DNA synthesis products (depicted in (A)), generated in extracts of transgenic loach larvae expressing non-functional and active forms of Pol ι (the data was derived from electropherogram (B) and calculations were performed using ImageQuant software). Standard deviation in all experiments was within 5%.

Figure 2

DNA synthesis in cell extracts using a substrate that contains a non-complementary nucleotide at the 3’-end of the primer. In all cases, quantitative parameters were determined from results of three to five independent experiments. (A) Alternative DNA synthesis products, generated in cell extracts with substrate No. 2. The frame highlights the nucleotides that are incorporated into substrate No. 2 during DNA synthesis. The dot above indicates the correct nucleotide incorporated in place of a mismatching nucleotide that was excised by exonucleases; (B) Electropherogram of DNA synthesis products generated in cell extracts of transgenic loach larvae that express either inactive (lane 1) or active Pol ι (lane 2) using substrate No. 2; (C) Electropherogram of DNA synthesis products that were generated in cell extracts of mice of the 129 line (lane 1) and the C57Bl line (lane 2); (D) Electropherogram of DNA synthesis products that were generated in cell extracts of loach larvae (lane 1) or in extracts of brain (lane 2) or testicular (lane 3) cells of adult fish; (E) Histogram representing the proportion of two alternative DNA synthesis products (sum of 18–21-mer) generated in tissue extracts of loach larvae and adult loaches using substrate No. 2 (the data was derived from electropherogram (D) and calculations were performed using ImageQuant software). Standard deviation in all experiments was within 5%.