FIG. 5.

Primed synthesis on the 11-nt oligoribonucleotide by wild-type primase and mutant proteins. (A) Sequence of the 11-nt oligoribonucleotide primer used for the reaction and G4oric DNA template. The 11-nt RNA oligonucleotide 5′-AGUAGGGACGG-3′ corresponded to the first 11 nucleotides (+1 to +11) of the pRNA product made by primase on the R199/G4oric template. (B) Primed synthesis on unlabeled 11-nt oligoribonucleotide, annealed to G4oric ssDNA template, by wild-type and mutant primase proteins in the presence of Mg²⁺ ions. CTP and [α-³²P]GTP were added to the reaction mixture as a source of nucleotides for the elongation. γ-³²P-labeled 11-nt RNA was used as a size marker. The control reaction contained no primase protein (lane 1). (C) Primed synthesis on unlabeled 11-nt oligoribonucleotide of the same sequence by wild-type and mutant primase proteins in the presence of Mn²⁺ ions. GTP and [α-³²P]CTP were added as a source of nucleotides for the elongation reaction. The control reaction contained no primase protein (lane 11).