Figure 5.

Functional Interaction of At PCNA2, Hs RP-A, and Hs PCNA with At DNA Pol λ during the Synthesis Opposite to 8-Oxo-G Damage.

(A) Protein gel blot analysis was performed using recombinant At PCNA2, At PCNA1, and At DNA pol λ proteins. Membrane A was spotted with PCNA1, PCNA2 (4 μg), or DNA pol λ (2μg); then membranes were overlaid with TBS buffer containing 0.1 mg/mL recombinant DNA pol λ and whose presence was revealed by anti-HsDNA pol λ antibodies. Membrane B was spotted with PCNA1, PCNA2, or BSA and then washed with TBS buffer and finally revealed by anti-Hs PCNA antibodies, as control experiment. The indicated amounts of BSA were spotted on both membranes as negative controls.

(B) Relative levels of dATP (white bars) and dCTP (gray bars) incorporation (expressed as percentage relative to the control in the absence of RP-A and PCNA) opposite to 8-oxo-G in the presence of different PCNA/RP-A combinations. Values are the mean of three independent biological replicates. Error bars represent ± sd values.

(C) Incorporation of dCTP (lanes 2 to 7) or dATP (lanes 8 to 13) in the absence (lanes 1 and 8) or in the presence (lanes 3 to 7 and 9 to 13) of 0.1 μM PCNA2 alone or in combination (lanes 4 to 7 and 10 to 13) with increasing amounts of Hs RP-A.