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bioRxiv · 10.1101/2022.02.09.479782

Multistep loading of PCNA onto DNA by RFC

Schrecker, M.·Castaneda, J. C.·Kumar, C.·Devbhandari, S.·Remus, D.·Hite, R. K.
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Abstract

The DNA sliding clamp proliferating cell nuclear antigen (PCNA) is an essential co-factor for many eukaryotic DNA metabolic enzymes. PCNA is loaded around DNA by the ATP-dependent clamp loader replication factor C (RFC), which acts at single-stranded/double-stranded DNA junctions harboring a recessed 3 end (3 ss/dsDNA junctions) and at DNA nicks. To illuminate the loading mechanism we have investigated the structure of RFC:PCNA bound to ATP{gamma}S and 3 ss/dsDNA junctions or nicked DNA using cryogenic electron microscopy. Unexpectedly, we observe open and closed PCNA conformations in the RFC:PCNA:DNA complex, revealing that PCNA can adopt an open, planar conformation that allows direct insertion of dsDNA, and indicating that PCNA ring-closure is not mechanistically coupled to ATP-hydrolysis. By resolving multiple DNA-bound states of RFC:PCNA we observe that partial melting facilitates lateral insertion into the central channel formed by RFC:PCNA. We also resolve the Rfc1 N-terminal domain and demonstrate that its single BRCT domain participates in coordinating DNA prior to insertion into the central RFC channel, which promotes PCNA loading on the lagging strand of replication forks in vitro. Combined, our data suggest a comprehensive and fundamentally revised model for the RFC-catalyzed loading of PCNA onto DNA.

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Schrecker, M., Castaneda, J. C., Kumar, C., Devbhandari, S., Remus, D., Hite, R. K.. 2022-02-09. Multistep loading of PCNA onto DNA by RFC. https://doi.org/10.1101/2022.02.09.479782

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