Figure 1.

Fluorometric assay to detect RNA-dependent HsPrimPol DNA primase/polymerase activity. (A) Fluorescence kinetics as a measure of de novo DNA synthesis by purified HsPrimPol (450 nM), in combination with different components as indicated. An increase in fluorescence as a function of time occurred only when dATP and dGTP (100 μM each), the GTCC template (1 μM; see Supplementary Table S1), and MnCl₂ (1 mM) were added. The use of the ATCC template (1 μM; Supplementary Table S1), or MgCl₂ as metal donor, did not render a measurable fluorescence signal. (B) Fluorescence kinetics using the optimal conditions described in A, but comparing GTCC and GUCC templates (see Supplementary Table S1). A negative control assay using GTCC template in the absence of dNTPs is represented as a comparison. Note that the scale of arbitrary units in the y-axis is different with respect to A. (C) Representative electropherogram of a priming experiment carried out with purified HsPrimPol (450 nM) and 16 nM [γ-32P]ATP, 100 μM dGTP and either GTCC or GUCC as template. The position of the labeled ‘5′-AdG-3′ dimer is indicated. Detailed experimental conditions, as well as templates sequences are described in Materials and Methods.