Figure 3: TthPrimPol is a DNA primase that can be coupled to processive elongation by Φ29DNApol.

(a) Left panel: metal and sugar selectivity of TthPrimPol primase activity. 3′-GTCC-5′ oligonucleotide (1 μM) was used as a preferred template. Labelled nucleotide [γ-³²P] ATP or [α-³²P] dATP nM) were alternatively used as 5′-nucleotide and either unlabelled GTP or dGTP (10 μM) were tested as 3′-nucleotide to form the initiating dimer. Primer synthesis mediated by TthPrimPol (400 nM) was evaluated either with 1 mM MnCl₂ or 5 mM MgCl₂ at 55 °C during 60 min. Right panel: recognition of the priming site. The assay was as in the left panel, but using templates differing in the base preceding the primase initiation site (…XTCC…) and the indicated metal and nucleotides. (b) TthPrimPol-mediated DNA primer synthesis at 30 °C occurs at multiple sites on a heterogeneous ssDNA template. TthPrimPol (100 nM) was able to generate DNA primers on circular M13mp18 ssDNA (5 ng μl⁻¹), when using four alternative combinations of dNTPs, implying that initiation occurred at multiple sites. In all cases, [α-³²P] dGTP (16 nM) was provided to label the nascent primers, combined with either dATP, dCTP, dGTP or dTTP (1 μM), in the presence of 10 mM MgCl₂ at 30 °C during 20 min. (c) To evaluate the processivity of primer synthesis by TthPrimPol, we used heparin as a competitor. TthPrimPol (10 nM) was preincubated for 5 min on ice, either in the absence/presence of heparin (1 ng μl⁻¹). Subsequently, the reaction was complemented with M13mp18 ssDNA (5 ng μl⁻¹), dATP, dCTP and dTTP (10 μM each), [α-³²P] dGTP (16 nM; 3,000 Ci mmol⁻¹) and heparin (1 ng μl⁻¹) when indicated and the incubation was maintained for 10 min at 30 °C, and processed as described. (d) TthPrimPol-synthesized DNA primers are efficiently extended by Φ29DNApol. The contribution of each enzyme was assayed in two consecutive stages (pulse and chase), as indicated in the scheme. The pulse demonstrated the synthesis of primers with a mean size of 7–9 nt (left panel). During chase (right panel), Φ29DNApol generated high-molecular-weight primer-elongated products (detected at the top of the gel) and some degradation products (evidenced at the bottom).