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

Abstract

Clamp loaders place circular sliding clamp proteins onto DNA so that clamp-binding partner proteins can synthesize, scan, and repair the genome. DNA with nicks or small single-stranded gaps are common clamp-loading targets in DNA repair, yet these substrates would be sterically blocked given the known mechanism for binding of primer-template DNA. Here, we report the discovery of a second DNA binding site in the yeast clamp loader Replication Factor C (RFC) that aids in binding to nicked or gapped DNA. This DNA binding site is on the external surface and is only accessible in the open conformation of RFC. Initial DNA binding at this site thus provides access to the primary DNA binding site in the central chamber. Furthermore, we identify that this site can partially unwind DNA to create an extended single-stranded gap for DNA binding in RFC's central chamber and subsequent ATPase activation. Finally, we show that deletion of the BRCT domain, a major component of the external DNA binding site, results in defective yeast growth in the presence of DNA damage where nicked or gapped DNA intermediates occur. We propose that RFC’s external DNA binding site acts to enhance DNA binding and clamp loading, particularly at DNA architectures typically found in DNA repair.

Unexpected new insights into DNA clamp loaders

“…[31,32,50] Consistent with a role in gap repair, the recent cryoEM data also reveal that RFC can even unwind dsDNA at both the internal and external DNA binding sites. [31,50] There appear to be two separation pins(for 3′ [Trp638 and Phe582] and 5′ [His556 and His659] ends) that may coordinate with each other at a nick to unwind up to five nucleotides of duplex DNA for clamp loading. [24] RFC mediated DNA unwinding at a nick is not driven by ATP hydrolysis but through DNA binding energy, and PCNA loading is within a few fold of the same efficiency as being loaded at a nick or gap compared to a 3′ primer template junction.…”

“…[ 31 , 32 , 50 ] Consistent with a role in gap repair, the recent cryoEM data also reveal that RFC can even unwind dsDNA at both the internal and external DNA binding sites. [ 31 , 50 ] There appear to be two separation pins(for 3′ [Trp638 and Phe582] and 5′ [His556 and His659] ends) that may coordinate with each other at a nick to unwind up to five nucleotides of duplex DNA for clamp loading. [ 24 ]…”

“…[24] RFC mediated DNA unwinding at a nick is not driven by ATP hydrolysis but through DNA binding energy, and PCNA loading is within a few fold of the same efficiency as being loaded at a nick or gap compared to a 3′ primer template junction. [32,50] Moreover, the intrinsic unwinding activity of RFC allows it to load PCNA at a variety of DNA structures. It seems possible that similar unwinding activities may facilitate other RLCs to function on particular DNA structures that are specific to them.…”

“…The 5′ DNA site of Rfc1 is not required for cell viability but mutations or deletions of the BRCT result in defective gap repair. [ 49 , 50 , 51 ] The possibility that RFC binds a DNA gap, via the BRCT region's ability to bind a recessed 5′ end, along with the 3′ primer terminus in the central chamber of the entire clamp loader, became obvious from the external DNA binding site in the Rad24‐RFC−9‐1‐1 structures. [ 45 , 46 ] Recent structures from three different labs have shown that this is indeed the case as illustrated in Figure 5 .…”

“…[47,48] The 5′ DNA site of Rfc1 is not required for cell viability but mutations or deletions of the BRCT result in defective gap repair. [49][50][51] The possibility that RFC binds a DNA gap, via the BRCT region's ability to bind a recessed 5′ end, along with the 3′ primer terminus in the . [50] central chamber of the entire clamp loader, became obvious from the external DNA binding site in the Rad24-RFC−9-1-1 structures.…”