Fig. 4: Gap-filling activities of Loop 3 mutants.
From: Molecular basis for DNA repair synthesis on short gaps by mycobacterial Primase-Polymerase C
a Short gap-filling activities of selected Prim–PolC mutations. In primer extension assays, 30 nM of 5′-fluorescein-labelled 36-mer containing a single-stranded 1-nt gap (top) or 3-nt gap (bottom) (with phosphorylation of the 5′ end of the downstream strand) was extended by Prim–PolC (300 nM) in the presence of a 250 μM rNTP mix for 1, 5 and 15 min at 37 °C. Control (C) lane contains no protein. b, c Quantification of the gap-filling assay (quantification of panel a). d Gap-filling activity of Prim–PolC (WT) on an abasic (template −2 position) 3-nt gap substrate. In primer extension assays, 30 nM of 5′-fluorescein-labelled 36-mer containing different single-stranded gaps, with phosphorylation of the 5′ end, was extended by Prim–PolC (30 nM) in the presence of a 250 μM rNTP mix for 1, 5 and 15 min at 37 °C. Control (C) lane contains no protein. e Quantification of Prim–PolC primer extension on an abasic 3-nt gap substrate (quantification of panel e). Data shown (b, c and e) are representative of the mean of at least three individual experiments, and error bars show the standard deviation, and dotplots show the corresponding data points.
