Single-Molecule Imaging of PAF15-PCNA using DNA Skybridge

Abstract

Proliferating cell nuclear antigen (PCNA) behaves as a sliding clamp that increases the processivity of eukaryotic and archea DNA polymerases through a physical interaction. Upon DNA damage during DNA replication, PCNA-associated factor 15 (PAF15) in complex with PCNA plays an important role in switching from a replicative polymerase to a translesion synthesis polymerase that is capable of bypassing nucleotide-lesions. Using the DNA skybridge that is a surface-condition independent and high-throughput single-molecule optical imaging platform, we monitored the diffusive motion of the PCNA-PAF15 complex on DNA molecules. We found that only one PAF15 molecule is associated with PCNA at physiological salt conditions, which results in slowing the diffusion of PCNA by up to 20 times and significantly stabilizing PCNA on DNA. The PCNA drag is likely to facilitate the switching of DNA polymerases. Taken together with the previous co-crystal structure of PCNA with a PAF15 fragment, our results propose a PAF15-PCNA conformation where the central domain of PAF15 interacts with the front face of PCNA and its N-terminal domain passes inside the PCNA ring.