Figure 4

AtPrimPol is a TLS DNA polymerase, (A) time course reaction from 10 to 40 min showing primer extension by wild-type AtPrimPol, primer extension corresponds to 27 nt, one nucleotide shorter than full extension, whereas AtPolIB exo⁻ extends to 28 nt and one nucleotide beyond. For 8-oxoG, polymerization products were observed from 5 to 40 min. For 5R and 5S thymine glycol isomer, PrimPol full-length was capable to extend up to 30, whereas AtPolIB exo-generated 30 nts full extension and 31 nts, but also displayed nucleotide incorporation product of 17 nt. For CPD and 6–4 PP, wild-type AtPrimPol showed a weak intermediate DNA product from 10 to 20 min (B) A single nucleotide insertion reactions were performed using 10 nM of dsDNA substrate (AP site, 8-oxoG, 5R-Tg or 5S-Tg) mixed with 200 nM of AtPrimPol. Individual reactions were initiated by addition of individual nucleotides and stopped at 10 min. For AP site, dAMP, dTMP and dGMP were incorporated with similar preference (lane 2, 3 and 4); for 8-oxoG, dAMP and dCMP were inserted preferentially (lane 7 and 10); for 5R-Tg and 5S-Tg isomer, wild-type PrimPol inserted randomly A, T, G or C, with a slight preference by dAMP and dCMP (lane 12 to 15 and 17 to 20). (C) E. coli BL21 (DE3) strain overexpressing a pET19:eGFP construct in which the chromophore sequence was replaced an A(AP site)G codon-like, and replicated by AtPolIA or AtPrimPol (upper panel), in the bottom panel, the template harbors a A(AP site)TG codon-like (Fig. S6C, D). Red arrows indicate overexpressing GFP colonies, white arrows a colony overexpressing nonfunctional GFP and yellow arrows indicates an emerald colony. Graphic of the relative percentage of white, emerald, and green colonies counted by triplicate. Original data used to compose this figure is present in Fig. S10.