The reactions included DNA substrates, cell extracts of the strains producing human Pol i, Mn2+ ions and various combinations of deoxynucleotides. Extracts of strains with empty vectors served as a negative control. For each extract, five different conditions have been used: 1) reaction with all four deoxynucleotides, 2) same but with dGTP omitted, 3) with only dGTP and dATP present, 4) with only dATP, and 5) with only dGTP. When none of the exogenous dNTPs were present, the synthesis was extremely weak and same faint bands corresponding to the correct BYL719 incorporation of dATP at position +1 were detected for the extract of yeast producing Pol i and the control extract, suggesting that the concentration of dNTPs in the extracts is low and does not support DNA synthesis under our conditions. The addition of a high concentration of exogenous dNTPs led to a strong stimulation of DNA synthesis. The misGvA activity was clearly seen for yeast extracts producing Pol i with all four dNTPs and when dATP and dGTP were present. In these experiments we compared two different oligonucleotide templates and, in addition to a negative control, we have used a positive control with extract of the strain producing human Pol g. The left panel of Fig. 2B represents primer extension reactions with template 1. Under our experimental conditions, crude cell extracts from the control strain with the vector alone possessed almost undetectable exonuclease activity and had moderate DNA polymerase activity with all four dNTPs. When dGTP was omitted from the reaction, the synthesis was halted almost exclusively at position 18, likely representing predominant dATP incorporation opposite template T. In the presence of dATP and dGTP, single bands are observed with a major termination site at position 21, when missing dTTP has to be inserted. In addition, the synthesis proceeded as for lane 1 until position 21, because only two nucleotides were required to copy the template. When dATP was present alone, the synthesis was stopped at position 18. The reaction with only dGTP in the absence of dATP was inefficient judging by a Tasocitinib higher percent of unutilized primer in line five for the control extract. The band with G at position 19 and minor bands at 18-position could be explained by the better extension of products driven by residual dNTPs in cell extracts when dGTP was in great excess. It is also possible that dGTP was incorporated by a template slippage mechanism, when the misaligned template guided incorporation of dGTP opposite +2 ����C���� in the template and, after immediate realignment. Next, dGTP opposite the same +2 ����C���� was incorporated. The dGTP incorporation in the 18 or 19 position did not occur when dATP was present in reactions. The pattern of band changes drastically when we used the extract from cells producing Pol i. These extracts possessed elevated DNA polymerase activity, as indicated by the small quantity of non-elongated primer, position 17. Apparently, most of this synthesis was due to Pol i. Most termination falls on the short elongation products, so that the bands at position 18 and 19 are more intense than with control extracts.