Figure 6.

End joining of a substrate containing a thymine glycol base. (A) Schematic of the internally labeled (asterisk) substrate, showing proposed mechanisms of formation of products giving 43-, 42- and 35-base BstXI/AvaI fragments. The italicized T is the site of thymine glycol substitution. Bolded nucleotides indicate gap filling. The 35-base product is shown as arising by 3′→5′ resection, but it could in principle be generated by 5′→3′ resection as well. (B) Unmodified or thymine glycol-substituted substrate was incubated for 6 h with X4L4-supplemented HeLa nuclear extracts that had been immunodepleted of polλ or mock depleted. Polλ or polμ (70 ng) was added as indicated. (C) Same as (B), except extracts were not immunodepleted, polλ was added to all samples and samples contained either ddCTP or ddTTP in place of normal dNTPs as indicated. (D) Quantitative data from (B) and similar experiments; the abundance of each product is normalized to the total of all head-to-tail end-joining products in the sample with the unmodified substrate (‘T’) and added polλ; ‘T-glycol’ = thymine glycol-substituted substrate. Error bars show mean and SE of 3–4 experiments. (E) Similar quantitation of data from (C) and a replicate experiment. Error bars show the range of values obtained in the two experiments.