FIG. 3.

pol μ ribonucleotide incorporation during end joining. (A) DNA-DNA, nicked DNA substrate; RNA-DNA, nicked DNA substrate with a single ribonucleotide at the 3′ terminus of the upstream strand. Nick ligation activity is shown as nanomoles of product per second per unit of ligase. The means of three replicate experiments are shown. The standard deviation was less than 10% of the mean for all substrates tested. (B) The 300-bp DNA end-joining substrate, aligned as required to form head-to-head ligation products as generated in panels C and D. (C) A 20 nM concentration of DNA substrate as in panel B was present in all reaction mixtures. Purified X4-LIV (50 nM) and DNA-PK (25 nM Ku plus 25 nM DNA-PKcs) were added as indicated (+). pol μ (μ; 25 nM) or pol β (β; 10, 25, or 50 nM [lanes 8 to 10, respectively]) was added as noted. All, 25 μM each dNTP; −T, 25 μM each dATP, dCTP, and dGTP; T, 25 μM dTTP only; S, substrate; P, ligation product. (D) All reactions as in panel C, lane 7, except that 25 μM dTTP (T), 500 μM rUTP (U), or 25 μM dTTP plus 500 μM rUTP (T+U) was included as noted. Following an in vitro end-joining reaction, samples were treated with alkali as indicated (+).