Cryo-EM structures reveal that RFC recognizes both the 3’- and 5’-DNA ends to load PCNA onto gaps for DNA repair

Abstract

RFC uses ATP to assemble PCNA onto primed sites for replicative DNA polymerases delta and epsilon;. The RFC pentamer forms a central chamber that binds a 3 primed DNA junction to load PCNA onto DNA during replication. We show here five structures revealing a novel second DNA binding site in RFC that binds a 5 prime junction. This 5 prime DNA site is located in the N-terminal BRCT domain and AAA module of the large Rfc1 subunit. It appears that 5 prime DNA binds after 3 prime DNA binding to RFC. Our structures reveal ideal binding to a 7-nt gap, which includes 2 bp unwound by the clamp loader. Biochemical studies show enhanced binding to 5 and 10 nucleotide gaps, consistent with the structural results. We propose that the 5 prime DNA site facilitates RFC PCNA loading activity at a DNA damage-induced gap to recruit gap-filling polymerases. These findings are consistent with genetic studies showing that base excision repair of gaps greater than 1 base requires PCNA and involves the 5 prime DNA binding domain of Rfc1. We further observe that a 5 prime site facilitates PCNA loading at an RPA coated 50-nt gap, suggesting a potential role of the RFC 5 prime DNA site at longer gaps including during lagging strand DNA synthesis.

A second DNA binding site on RFC facilitates clamp loading at gapped or nicked DNA

“…Thus, Rfc1 has two separation pins, one in the central chamber and a newly discovered one on the external DNA binding site. This external separation pin is remarkably similar to that used by Rad24-RLC when it binds DNA with a recessed 5' end (Castaneda et al, 2021;Zheng et al, 2021) (see discussion for more details).…”

“…with a recessed 5' end) (Ellison and Stillman, 2003;Majka and Burgers, 2004). Recent structures of Rad24-RLC bound to 9-1-1 and 5'-recessed DNA have revealed the surprising finding that the duplex DNA region is bound on the external surface of the A-subunit Rad24, with the 3' ssDNA overhang snaking through the A-gate into the central chamber (Figure 6A) (Zheng et al 2021;Castaneda et al 2021).…”

“…Moreover, non-classical clamp loaders called RFC-Like Complexes (RLCs), such as Rad24-RLC and Elg1-RLC utilize DNA structures with recessed 5' ends or fully dsDNA, respectively (Kubota et al, 2015;Majka and Burgers, 2004;Majka et al, 2006). Recent structures of Rad24-RLC show that the 5' recessed DNA is surprisingly accommodated on a second DNA binding site on the "shoulder" of Rad24 (Castaneda et al 2021;Zheng et al 2021). However, the structures of RFC and Rad24-RLC do not indicate how gapped or nicked DNA can bind to RFC.…”

Unexpected new insights into DNA clamp loaders

“…[ 46 ] (b) Domain structure of Rfc1 adapted from figure 2 of Zheng et al. [ 32 ] (C) Domain structure of Rfc24 adapted from figure 2 of Zheng et al. [ 46 ]…”

“…However, Rad24 also contains a C-terminal coiled-coil domain known to bind RPA, which may provide it with additional functionality. Adapted from figure 1a of Zheng et al [46] (b) Domain structure of Rfc1 adapted from figure 2 of Zheng et al [32] (C) Domain structure of Rfc24 adapted from figure 2 of Zheng et al [46] all three domains of life [7] and are summarized in the illustration of Figure 1B.…”