FIGURE 5.

DNA pol β favors smaller gap size than DNA pol λ in TLS of an AP site in the presence of DNA pol ϵ. Experiments were performed with templates shown in Table 1a. The polymerases and the DNA substrates used are indicated at the top. ss stands for template-primer with no oligonucleotides hybridized downstream from the AP site, whereas 2 and 4 indicate the length of gap regions between the AP site and the oligonucleotide hybridized downstream. Assays were carried out as described under “Experimental Procedures.” A, lane 1, no polymerase present. Lane 2, reaction incubated for 35 min with 0.025 pmol of pol ϵ. Lanes 3 and 4, reactions incubated with 0.25 pmol of pol λ for 5 and 10 min, respectively. Lanes 5 and 6, reactions incubated with 0.25 pmol of pol β for 5 and 10 min, respectively. Lanes 7 and 8, reactions were incubated with 0.025 pmol of pol ϵ for 30 min, then 0.25 pmol of pol λ were added and the incubation continued for 5 and 10 min, respectively. Lanes 9–12: as for lanes 7 and 8. Lanes 13 and 14, reactions were incubated with 0.025 pmol of pol ϵ for 30 min, then 0.25 pmol of pol β were added and the incubation continued for 5 and 10 min, respectively. Lanes 15–18: as for lanes 13 and 14. The positions of the primer and of the AP site are indicated. B, quantification of the data from A with pol λ, expressed as percentage of TLS calculated as described under “Experimental Procedures.” White bar, pol ϵ alone. Light gray bars, incubation with pol ϵ and then pol λ added for 5 min. Dark gray bars, incubation with pol ϵ and then pol λ added for 10 min. C, quantification of the data from A with pol β, expressed as percentage of TLS and calculated as described under “Experimental Procedures.” Light gray bars, incubation with pol ϵ and pol β added for 5 min. Dark gray bars, incubation with pol ϵ and pol β added for 10 min.