scholarly journals Rad24-RFC loads the 9-1-1 clamp by inserting DNA from the top of a wide-open ring, opposite the mechanism of RFC/PCNA

2021 ◽  
Author(s):  
Fengwei Zheng ◽  
Roxana E. Georgescu ◽  
Nina Y. Yao ◽  
Michael E. O’Donnell ◽  
Huilin Li

ABSTRACTIn response to DNA damage, the ring-shaped 9-1-1 clamp is loaded onto 5’ recessed DNA to arrest the cell cycle and activate the DNA damage checkpoint. The 9-1-1 clamp is a heterotrimeric ring that is loaded in S. cerevisiae by Rad24-RFC, an alternative clamp loader in which Rad24 replaces the Rfc1 subunit in the RFC1-5 clamp loader of PCNA. Unlike RFC that loads the PCNA ring onto a 3’-ss/ds DNA junction, Rad24-RFC loads the 9-1-1 ring onto a 5’-ss/ds DNA junction, a consequence of DNA damage. The underlying 9-1-1 clamp loading mechanism has been a mystery. Here we report two 3.2-Å cryo-EM structures of Rad24-RFC bound to DNA and either a closed or 27 Å open 9-1-1 clamp. The structures reveal a completely unexpected mechanism by which a clamp can be loaded onto DNA. The Rad24 subunit specifically recognizes the 5’-DNA junction and holds ds DNA outside the clamp loader and above the plane of the 9-1-1 ring, rather than holding DNA inside and below the clamp as in RFC. The 3’ ssDNA overhang is required to obtain the structure, and thus confers a second DNA binding site. The bipartite DNA binding by Rad24-RFC suggests that ssDNA may be flipped into the open 9-1-1 ring, similar to ORC-Cdc6 that loads the Mcm2-7 ring on DNA. We propose that entry of ssDNA through the 9-1-1 ring triggers the ATP hydrolysis and release of the Rad24-RFC. The key DNA binding residues are conserved in higher eukaryotes, and thus the 9-1-1 clamp loading mechanism likely generalizes.

1999 ◽  
Vol 19 (11) ◽  
pp. 7501-7510 ◽  
Author(s):  
Andrei L. Okorokov ◽  
Jo Milner

ABSTRACT Interaction with DNA is essential for the tumor suppressor functions of p53. We now show, for the first time, that the interaction of p53 with DNA can be stabilized by small molecules, such as ADP and dADP. Our results also indicate an ATP/ADP molecular switch mechanism which determines the off-on states for p53-DNA binding. This ATP/ADP molecular switch requires dimer-dimer interaction of the p53 tetramer. Dissociation of p53-DNA complexes by ATP is independent of ATP hydrolysis. Low-level ATPase activity is nonetheless associated with ATP-p53 interaction and may serve to regenerate ADP-p53, thus recycling the high-affinity DNA binding form of p53. The ATP/ADP regulatory mechanism applies to two distinct types of p53 interaction with DNA, namely, sequence-specific DNA binding (via the core domain of the p53 protein) and binding to sites of DNA damage (via the C-terminal domain). Further studies indicate that ADP not only stabilizes p53-DNA complexes but also renders the complexes susceptible to dissociation by specific p53 binding proteins. We propose a model in which the DNA binding functions of p53 are regulated by an ATP/ADP molecular switch, and we suggest that this mechanism may function during the cellular response to DNA damage.


2021 ◽  
Author(s):  
Juan C Castaneda ◽  
Marina Schrecker ◽  
Dirk Remus ◽  
Richard K Hite

5' single-stranded/double-stranded DNA serve as loading sites for the checkpoint clamp, 9-1-1, which mediates activation of the apical checkpoint kinase, ATRMec1. However, the basis for 9-1-1's recruitment to 5' junctions is unclear. Here, we present structures of the yeast checkpoint clamp loader, Rad24-RFC, in complex with 9-1-1 and a 5' junction and in a post-ATP-hydrolysis state. Unexpectedly, 9-1-1 adopts both closed and planar open states in the presence of Rad24-RFC and DNA. Moreover, Rad24-RFC associates with the DNA junction in the opposite orientation of processivity clamp loaders with Rad24 exclusively coordinating the double-stranded region. ATP hydrolysis stimulates conformational changes in Rad24-RFC, leading to disengagement of DNA-loaded 9-1-1. Together, these structures explain 9-1-1's recruitment to 5' junctions and reveal new principles of sliding clamp loading.


2004 ◽  
Vol 279 (20) ◽  
pp. 20921-20926 ◽  
Author(s):  
Jerzy Majka ◽  
Brian Y. Chung ◽  
Peter M. J. Burgers

2018 ◽  
Author(s):  
Roberto Vazquez Nunez ◽  
Laura B. Ruiz Avila ◽  
Stephan Gruber

SummaryMulti-subunit SMC ATPase complexes translocate on chromosomal DNA. They control chromosome structure and DNA topology, presumably by acting as DNA extrusion motors. The SMC-kleisin ring entraps chromosomal DNA. The ring lumen is strongly reduced in size by alignment of the SMC arms and upon ATP binding is divided in two by engagement of SMC head domains. Here, we provide evidence for DNA binding in the SMC compartment and chromosome entrapment in the Kleisin compartment of B. subtilis Smc/ScpAB. We show that DNA binding at the Smc hinge is dispensable and identify an essential DNA binding site at engaged heads which faces the S compartment. Mutations interfering with DNA binding do not prevent ATP hydrolysis but block DNA translocation by Smc/ScpAB. Our findings are consistent with the notion that Smc/DNA contacts stabilize looped DNA segments in the S compartment, while the base of a chromosomal DNA loop is enclosed in the K compartment. Transfer of DNA double helices between S and K compartments may support DNA translocation.


2001 ◽  
Vol 21 (11) ◽  
pp. 3725-3737 ◽  
Author(s):  
Hee-Sook Kim ◽  
Steven J. Brill

ABSTRACT The large subunit of replication protein A (Rpa1) consists of three single-stranded DNA binding domains and an N-terminal domain (Rpa1N) of unknown function. To determine the essential role of this domain we searched for mutations that require wild-type Rpa1N for viability in yeast. A mutation in RFC4, encoding a small subunit of replication factor C (RFC), was found to display allele-specific interactions with mutations in the gene encoding Rpa1 (RFA1). Mutations that map to Rpa1N and confer sensitivity to the DNA synthesis inhibitor hydroxyurea, such asrfa1-t11, are lethal in combination withrfc4-2. The rfc4-2 mutant itself is sensitive to hydroxyurea, and like rfc2 and rfc5 strains, it exhibits defects in the DNA replication block and intra-S checkpoints. RFC4 and the DNA damage checkpoint geneRAD24 were found to be epistatic with respect to DNA damage sensitivity. We show that the rfc4-2 mutant is defective in the G1/S DNA damage checkpoint response and that both therfc4-2 and rfa1-t11 strains are defective in the G2/M DNA damage checkpoint. Thus, in addition to its essential role as part of the clamp loader in DNA replication, Rfc4 plays a role as a sensor in multiple DNA checkpoint pathways. Our results suggest that a physical interaction between Rfc4 and Rpa1N is required for both roles.


2009 ◽  
Vol 34 (6) ◽  
pp. 735-745 ◽  
Author(s):  
Andrew S. Doré ◽  
Mairi L. Kilkenny ◽  
Neil J. Rzechorzek ◽  
Laurence H. Pearl

Biochemistry ◽  
2009 ◽  
Vol 48 (36) ◽  
pp. 8516-8527 ◽  
Author(s):  
Stephen G. Anderson ◽  
Jennifer A. Thompson ◽  
Christopher O. Paschall ◽  
Mike O’Donnell ◽  
Linda B. Bloom

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