Extended Data Fig. 4: Priming requirements and mismatch tolerance of ORF2p core.
From: Structures, functions and adaptations of the human LINE-1 ORF2 protein
a, Comparison of 10 nt vs 20 nt DNA primers reveals little difference in efficiency of formation of products, including larger template jumping/switching products (##). b, ORF2p performs DNA synthesis with 5–10 nt DNA primers, although 5 nt and, to a lesser extent 6 nt, are slightly less efficiently used. As seen consistently above, RNA templates are slightly less efficient than DNA. Higher concentrations of ORF2p core result in higher activity in all conditions and more template jumping/switching products. Scanned gel images are cropped and corrected for distortion artifacts with contrast uniformly increased to facilitate the visualization of minor products. (* indicates Cy5 label, all panels). c, RNA synthesis is strongly selected against, as indicated by nucleotide (dNTP or NTP) incorporation activity of LINE-1 RT on DNA or RNA using a DNA primer. Denaturing PAGE migration pattern of the reaction products generated after 5 min of dNTP or NTP incorporation along DNA and RNA templates using 20-nt primers. d, Priming activity of ORF2p and HIV-1 with one or two terminal mismatches; two enzyme preps of HIV-1 RT are compared to ORF2p, and additional unextended substrates are shown. L1 tolerates all terminal mismatches against an A template to some extent, as well as some penultimate mismatches; A:G is inefficient. In contrast, HIV-1 is less tolerant of A:A and A:G terminal and U:A and U:G penultimate mismatches; n = 1 (LINE-1) and n = 2 (HIV-1) points quantified from 2 independent experiments.
