Figure 5.

De Novo DNA Synthesis by piPolB

(A) Primer synthesis by piPolB. M13 DNA was incubated with either dNTPs or NTPs and wild-type (WT) piPolB or the polymerase- (D368A) or exonuclease- (D59A/E61A) deficient variants. Detected products are labeled with [γ³²P]ATP (1 μCi) that only could be incorporated in the 5′ position of the newly synthetized primers. A [γ³²P]ATP-(dGMP)n ladder was used a size marker (lane M).

(B) Insertion preference for the first steps of DNA primer synthesis by exonuclease-deficient piPolB. The assay was performed as in (A) but with each dNTP provided independently or in the indicated combinations. Reactions were triggered with either 10 mM MgCl₂ or 1 mM MnCl₂ and resolved in high-resolution 8 M urea-20% PAGE.