To confirm these results, a polymerization assay based on the work of Koepsell et al.7 in which the 37-mer single stranded DNA template 5′-CAG(AC)12ACTACACACA-3′ (transcription initiation start site at T, coordinate 30, underlined) was used. The known inhibitor of DnaG primase activity, doxorubicin,4 was included as a positive control for these studies (Figure 1b, Lane 1). The complete in vitro reaction was supplemented with an incremental series of TG concentrations. Polymerization products labeled by inclusion of [α-32P]UTP in the reaction mix were fractionated by PAGE and analyzed by autoradiography.8 The results, Figure 1b, lanes 2 and 3, show the expected 30-mer RNA primer that is formed in the complete uninhibited reaction. Results shown in lanes 4, 5 and 6, indicate that TG inhibits RNA primer formation in a TG-dependent manner with nearly complete inhibition of 30-mer transcript formation at 20 μM, near the MIC. Lanes 6 and 7 show the reappearance of transcript formation and a shift of the transcription product length to 60 nt. The 60-mer product may correspond to RNA formed by extension of the 37-mer template beginning at its 3′OH, and it is similar to the overlong product seen in earlier reports.9 We postulate that the 60-nt product is an RNA-DNA heteroduplex hairpin loop in which 5′-TACACACA-3′ forms the loop portion by association of the 5′T with the 3′A. Lane 8 shows the products formed by DnaG in the presence of 100 μM TG–complete abolition of 30-mer synthesis and marked reduction in 60-mer synthesis.

To compare the specificity of DnaG with that of other polymerases, four additional enzymes were tested, namely, Escherichia coli DNA-dependent RNA polymerase and E. coli PolA (NEB, Ipswich, MA, USA); T7 DNA-dependent RNA polymerase (Fermentas, Waltham, MA, USA); and MuLV reverse transcriptase (Thermo Fisher, Fitchburg, WI, USA). The DNA templates used for the four enzymes were, calf thymus DNA, DNAase-treated calf thymus DNA, PCR-amplified T7 polymerase promoter sequence, and Bacillus. subtilis total RNA, respectively. Polymerase reactions were performed based on protocols provided by the vendor.

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