Abstract
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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Acknowledgements
We thank Dr. Rodger Woodgate (National Institutes of Health, Bethesda, USA) for providing plasmid encoding human Pol ι and Dr. Andrey Kulbachinskiy (Institute of Molecular Genetics, Moscow, Russia) for the careful review of this paper. This study was supported by the Program of fundamental research of the Russian Academy of Sciences “Biodiversity”, the Federal Targeted Program “Scientific and scientific-pedagogical personnel of the innovative Russia” 2009-2013 (state contract №П335) and Russian Foundation for Basic Research Program.
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Fig. S1
Supplementary material 1 “misGvA” activity in loach embryos demonstrating abnormalities in development. DNA-polymerase activity was analyzed in the extracts of loach embryos injected with pCI-neo, pPINC, pEGFP or pEGFP-POLI plasmids on 2–5 dpf. Embryos with developmental abnormalities designated as “A”, normally developing embryos – “N”. (TIFF 3893 kb)
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Makarova, I.V., Kazakov, A.A., Makarova, A.V. et al. Transient expression and activity of human DNA polymerase iota in loach embryos. Biotechnol Lett 34, 205–212 (2012). https://doi.org/10.1007/s10529-011-0764-8
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DOI: https://doi.org/10.1007/s10529-011-0764-8