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

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.

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

“…We have previously demonstrated that the misincorporation activity of Pol ι in intact loach could not be detected at any stage of development [ 21 ]. After injection of human Pol ι encoding gene into loach embryos, we have observed the appearance of activity of this enzyme in cellular extracts of GFP-expressing larvae ( Figure 1 B) by using the radiolabeled primer extension method [ 25 , 26 ].…”

“…DNA fragments corresponding to human Pol ι cDNA along with 60 bp 5’-untranslated region sequence were inserted between the NcoI and BamHI sites of pEGFP-C1 vector (Clontech, Montain View, CA, USA), downstream of a cytomegalovirus (CMV) promoter, to generate the fusion protein eGFP-POLI (pEGFP-POLI vector), as described previously [ 21 ]. The plasmid containing cDNA encoding an inactive mutant form of the Pol ι protein (D126A-E127A) fused with eGFP was generated using site-specific mutagenesis of pEGFP-POLI vector by Evrogen (Moscow, Russia).…”

“…The plasmid containing cDNA encoding an inactive mutant form of the Pol ι protein (D126A-E127A) fused with eGFP was generated using site-specific mutagenesis of pEGFP-POLI vector by Evrogen (Moscow, Russia). To obtain transgenic 5–10 day loach larvae (Misgurnus fossilis L), the linearized recombinant DNAs encoding active or inactive forms of human Pol ι were injected into fertilized eggs at the germinal disc stage, as was previously described [ 21 ]. The expression of Pol ι in loach larvae was detected by Western blotting, and the activity of Pol ι was determined by primer extension reaction (the procedures and figures were presented in our previous work [ 21 ]).…”