FIGURE 7.

PCNA and RPA have no effect on gap size specificity of TLS of an AP site by DNA pol λ in presence of DNA pol ϵ. Experiments were performed with templates shown in Table 1a. The proteins 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 1, 2, and 4, 6, 8, 10, and 13 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, 1.2 pmol of PCNA, and 0.25 pmol of RPA. Lanes 3–10, the reactions were incubated for 30 min with 0.025 pmol of pol ϵ, 1.2 pmol of PCNA, and 0.25 pmol of RPA, and then 0.25 pmol of pol λ were added, and the incubation was continued for 5 min. The positions of the primer and of the AP site are indicated. B, quantification of data from Fig. 6A, expressed as percentage of TLS calculated as described under “Experimental Procedures.” White bar, reaction without pol λ. Gray bar, complete reaction.