Figure 4.

His¹⁶⁹ is essential for dimer synthesis and primer extension, Ser¹⁶⁷ and Lys²⁹⁷ are required to make dimers but not for primer extension, and Lys¹⁶⁵, Ser¹⁶⁷ and Lys²⁹⁷ are needed to form non-canonical extension products. (A) Dimer formation and further elongation by WT HsPrimPol or the indicated mutants (100 nM) using a template with the preferred priming site (3′ GTC) followed by a heterologous sequence, by sequentially adding [γ-³²P]ATP (16 nM) (γ-³²P is indicated as a red ball) as the 5′ nucleotide, and dGTP, dTTP and dCTP (100 μM) as 3′ incoming dNTPs, in the presence of 1 mM MnCl₂. (B) Elongation of a synthetic mini-primer (_3pAGT in blue with a blue box; 10 μM;) by WT HsPrimPol or the indicated mutants (100 nM) in the presence of dCTP, dTTP (10 μM), [α-³²P]dGTP (16 nM; in red), and 1 mM MnCl₂. Labeled dGMP insertion is indicated with a red ball. Non-canonical products are marked by asterisks.