8FS8

Structure of S. cerevisiae Rad24-RFC loading the 9-1-1 clamp onto a 5-nt gapped DNA (9-1-1 encircling fully bound DNA)

Summary for 8FS8

• Entry DOI: 10.2210/pdb8fs8/pdb
• EMDB information: 29417
• Descriptor: Checkpoint protein RAD24, Primer strand 1, Primer strand 2, ... (14 entities in total)
• Functional Keywords: dna damage repair, rad24-rfc, 9-1-1 clamp, dna clamp, alternative clamp loader, dna damage signaling, dna binding protein-dna complex, cell cycle-dna complex, cell cycle/dna
• Biological source: Saccharomyces cerevisiae (baker's yeast); More
• Total number of polymer chains: 11
• Total formula weight: 410421.77

Authors

Zheng, F.,Georgescu, R.,Yao, Y.N.,O'Donnell, M.E.,Li, H. (deposition date: 2023-01-09, release date: 2023-06-14, Last modification date: 2024-06-19)

Primary citation

Zheng, F.,Georgescu, R.E.,Yao, N.Y.,O'Donnell, M.E.,Li, H.
Structures of 9-1-1 DNA checkpoint clamp loading at gaps from start to finish and ramification to biology.
Biorxiv, 2023

PubMed Abstract: Recent structural studies show the Rad24-RFC loads the 9-1-1 checkpoint clamp onto a recessed 5' end by binding the 5' DNA on Rad24 at an external surface site and threading the 3' ssDNA into the well-established internal chamber and into 9-1-1. We find here that Rad24-RFC loads 9-1-1 onto DNA gaps in preference to a recessed 5' DNA end, thus presumably leaving 9-1-1 on a 3' ss/ds DNA after Rad24-RFC ejects from the 5' gap end and may explain reports of 9-1-1 directly functioning in DNA repair with various TLS polymerases, in addition to signaling the ATR kinase. To gain a deeper understanding of 9-1-1 loading at gaps we report high-resolution structures of Rad24-RFC during loading of 9-1-1 onto 10-nt and 5-nt gapped DNAs. At a 10-nt gap we captured five Rad24-RFC-9-1-1 loading intermediates in which the 9-1-1 DNA entry gate varies from fully open to fully closed around DNA using ATPγS, supporting the emerging view that ATP hydrolysis is not needed for clamp opening/closing, but instead for dissociation of the loader from the clamp encircling DNA. The structure of Rad24-RFC-9-1-1 at a 5-nt gap shows a 180° axially rotated 3'-dsDNA which orients the template strand to bridge the 3'- and 5'- junctions with a minimum 5-nt ssDNA. The structures reveal a unique loop on Rad24 that limits the length of dsDNA in the inner chamber, and inability to melt DNA ends unlike RFC, thereby explaining Rad24-RFC's preference for a preexisting ssDNA gap and suggesting a direct role in gap repair in addition to its checkpoint role.

PubMed: 37205533
DOI: 10.1101/2023.05.03.539266

Experimental method

ELECTRON MICROSCOPY (3.04 Å)