Figure 2.

Gap filling and accurate end joining of a DSB substrate bearing partially complementary overhangs. (A) Schematic of the site-specifically labeled (asterisk) DSB substrate showing accurate end joining by gap filling and ligation, resulting in a 43-base BstXI/AvaI fragment. All sequences read 5′→3′ in the top strand and 3′→5′ in the bottom strand. Bolded T indicates the filled-in base. (B) The substrate shown, bearing -ACA and -ATG 3′ overhangs, was incubated for 6 h with X4L4-supplemented HeLa extracts that had been immunodepleted of polλ or mock depleted. Polλ, polμ or pol-ch (70 ng) was also added as indicated. After incubation, samples were cut with BstXI and AvaI, and analyzed on denaturing gels. (C) Same as (B), except the reaction was performed in the presence of ddTTP instead of dTTP, and only some samples contained X4L4, as indicated. ‘ch*’ indicates 280 ng of pol-ch was added. (D) Quantitation of data from (B) and from a replicate experiment; error bars show the range of values obtained in the two experiments.