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

Abstract

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 baker's 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.

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

“…Pol i functions are still unclear thus necessitating the search for new methods and models for studies of Pol i expression, biochemical properties, functions, and regulation. Previously we introduced the ''misGvA'' method which could be applied as a simple approach to monitor Pol i activity and replication fidelity in various living cells and tissues types (Gening et al 2006;Kazakov et al 2008;Makarova et al 2011). The method does not require enzyme purification and allow detecting Pol i activity in the presence of accessory factors and possible posttranslational modifications.…”

“…The primer 5 0 -GGAAGAAGAAG TATGTT-3 0 and template 1 -5 0 -CCTTCTTCATT CTAACATACTTCTTCTTCC-3 0 were the same as in the works (Gening et al 2006;Zhang et al 2000). The template 2 -5 0 -CCTTCTTCATTGTAACATACTT CTTCTTCC-3 0 was described in work (Makarova et al 2011). The template 2 is similar to template 1, but contains G instead of C at position 19 of the 30-mer template (underlined).…”

“…Analysis of ''misGvA'' activity in loach embryos transiently expressing human POLI Previously we proposed a method of Pol i biochemical activity assay in extracts of cells based on a unique ability of Pol i to preferentially insert dGTP opposite T (''misGvA'' assay) (Gening et al 2006;Kazakov et al 2008;Makarova et al 2011). The principle of the method is shown in Fig.…”

“…2b). All experiments were also duplicated using DNA template 2 containing the substitution of nucleotide from C to G at 19-position to ensure that ''misGvA'' activity is not a result of dGTP incorporation by a template slippage mechanism (Makarova et al 2011). The results for templates 2 were same as for template 1 and are not shown.…”

“…ions with Mn 2? cations and applied it for testing activity of human Pol i produced in yeast (Makarova et al 2011).…”