scholarly journals Interplay of Clamp Loader Subunits in Opening the β Sliding Clamp ofEscherichia coliDNA Polymerase III Holoenzyme

2001 ◽  
Vol 276 (50) ◽  
pp. 47185-47194 ◽  
Author(s):  
Frank P. Leu ◽  
Mike O'Donnell
Keyword(s):  
Nuclear Antigen ◽  
Clamp Loader ◽  
Lower Efficiency ◽  
E Coli ◽  
Sliding Clamp ◽  
Polymerase Iii ◽  
Clamp Loading ◽  
Factor C ◽  
Opening And Closing ◽  
Proliferating Cell

TheEscherichia coliβ dimer is a ring-shaped protein that encircles DNA and acts as a sliding clamp to tether the replicase, DNA polymerase III holoenzyme, to DNA. The γ complex (γδδ′χψ) clamp loader couples ATP to the opening and closing of β in assembly of the ring onto DNA. These proteins are functionally and structurally conserved in all cells. The eukaryotic equivalents are the replication factor C (RFC) clamp loader and the proliferating cell nuclear antigen (PCNA) clamp. The δ subunit of theE. coliγ complex clamp loader is known to bind β and open it by parting one of the dimer interfaces. This study demonstrates that other subunits of γ complex also bind β, although weaker than δ. The γ subunit like δ, affects the opening of β, but with a lower efficiency than δ. The δ′ subunit regulates both γ and δ ring opening activities in a fashion that is modulated by ATP interaction with γ. The implications of these actions for the workings of theE. coliclamp loading machinery and for eukaryotic RFC and PCNA are discussed.

scholarly journals Structure of the human clamp loader reveals an autoinhibited conformation of a substrate-bound AAA+ switch

2020 ◽  
Vol 117 (38) ◽  
pp. 23571-23580 ◽  
Author(s):  
Christl Gaubitz ◽  
Xingchen Liu ◽  
Joseph Magrino ◽  
Nicholas P. Stone ◽  
Jacob Landeck ◽  
...  
Keyword(s):  
Active Sites ◽  
Mechanistic Explanation ◽  
Nuclear Antigen ◽  
Clamp Loader ◽  
Sliding Clamp ◽  
Disease Mutations ◽  
Clamp Loading ◽  
Clamp Loaders ◽  
Factor C ◽  
Aaa Atpases

DNA replication requires the sliding clamp, a ring-shaped protein complex that encircles DNA, where it acts as an essential cofactor for DNA polymerases and other proteins. The sliding clamp needs to be opened and installed onto DNA by a clamp loader ATPase of the AAA+ family. The human clamp loader replication factor C (RFC) and sliding clamp proliferating cell nuclear antigen (PCNA) are both essential and play critical roles in several diseases. Despite decades of study, no structure of human RFC has been resolved. Here, we report the structure of human RFC bound to PCNA by cryogenic electron microscopy to an overall resolution of ∼3.4 Å. The active sites of RFC are fully bound to adenosine 5′-triphosphate (ATP) analogs, which is expected to induce opening of the sliding clamp. However, we observe the complex in a conformation before PCNA opening, with the clamp loader ATPase modules forming an overtwisted spiral that is incapable of binding DNA or hydrolyzing ATP. The autoinhibited conformation observed here has many similarities to a previous yeast RFC:PCNA crystal structure, suggesting that eukaryotic clamp loaders adopt a similar autoinhibited state early on in clamp loading. Our results point to a “limited change/induced fit” mechanism in which the clamp first opens, followed by DNA binding, inducing opening of the loader to release autoinhibition. The proposed change from an overtwisted to an active conformation reveals an additional regulatory mechanism for AAA+ ATPases. Finally, our structural analysis of disease mutations leads to a mechanistic explanation for the role of RFC in human health.

scholarly journals Quality control mechanisms exclude incorrect polymerases from the eukaryotic replication fork

2017 ◽  
Vol 114 (4) ◽  
pp. 675-680 ◽  
Author(s):  
Grant D. Schauer ◽  
Michael E. O’Donnell
Keyword(s):  
Quality Control ◽  
Nuclear Antigen ◽  
Control Mechanisms ◽  
Clamp Loader ◽  
Release Process ◽  
Leading Strand ◽  
Polymerase Binding ◽  
Factor C ◽  
Proliferating Cell ◽  
Lagging Strand

The eukaryotic genome is primarily replicated by two DNA polymerases, Pol ε and Pol δ, that function on the leading and lagging strands, respectively. Previous studies have established recruitment mechanisms whereby Cdc45-Mcm2-7-GINS (CMG) helicase binds Pol ε and tethers it to the leading strand, and PCNA (proliferating cell nuclear antigen) binds tightly to Pol δ and recruits it to the lagging strand. The current report identifies quality control mechanisms that exclude the improper polymerase from a particular strand. We find that the replication factor C (RFC) clamp loader specifically inhibits Pol ε on the lagging strand, and CMG protects Pol ε against RFC inhibition on the leading strand. Previous studies show that Pol δ is slow and distributive with CMG on the leading strand. However, Saccharomyces cerevisiae Pol δ–PCNA is a rapid and processive enzyme, suggesting that CMG may bind and alter Pol δ activity or position it on the lagging strand. Measurements of polymerase binding to CMG demonstrate Pol ε binds CMG with a Kd value of 12 nM, but Pol δ binding CMG is undetectable. Pol δ, like bacterial replicases, undergoes collision release upon completing replication, and we propose Pol δ–PCNA collides with the slower CMG, and in the absence of a stabilizing Pol δ–CMG interaction, the collision release process is triggered, ejecting Pol δ on the leading strand. Hence, by eviction of incorrect polymerases at the fork, the clamp machinery directs quality control on the lagging strand and CMG enforces quality control on the leading strand.

scholarly journals Impact of Individual Proliferating Cell Nuclear Antigen-DNA Contacts on Clamp Loading and Function on DNA

2012 ◽  
Vol 287 (42) ◽  
pp. 35370-35381 ◽  
Author(s):  
Yayan Zhou ◽  
Manju M. Hingorani
Keyword(s):  
Steady State ◽  
Proliferating Cell Nuclear Antigen ◽  
Nuclear Antigen ◽  
Replication Factor ◽  
Terminal Domain ◽  
Clamp Loading ◽  
Factor C ◽  
Cationic Residues ◽  
Proliferating Cell

Ring-shaped clamp proteins encircle DNA and affect the work of many proteins, notably processive replication by DNA polymerases. Crystal structures of clamps show several cationic residues inside the ring, and in a co-crystal of Escherichia coli β clamp-DNA, they directly contact the tilted duplex passing through (Georgescu, R. E., Kim, S. S., Yurieva, O., Kuriyan, J., Kong, X. P., and O'Donnell, M. (2008) Structure of a sliding clamp on DNA. Cell 132, 43–54). To investigate the role of these contacts in reactions involving circular clamps, we examined single arginine/lysine mutants of Saccharomyces cerevisiae proliferating cell nuclear antigen (PCNA) in replication factor C (RFC)-catalyzed loading of the clamp onto primer template DNA (ptDNA). Previous kinetic analysis has shown that ptDNA entry inside an ATP-activated RFC-PCNA complex accelerates clamp opening and ATP hydrolysis, which is followed by slow PCNA closure around DNA and product dissociation. Here we directly measured multiple steps in the reaction (PCNA opening, ptDNA binding, PCNA closure, phosphate release, and complex dissociation) to determine whether mutation of PCNA residues Arg-14, Lys-20, Arg-80, Lys-146, Arg-149, or Lys-217 to alanine affects the reaction mechanism. Contrary to earlier steady state analysis of these mutants (McNally, R., Bowman, G. D., Goedken, E. R., O'Donnell, M., and Kuriyan, J. (2010) Analysis of the role of PCNA-DNA contacts during clamp loading. BMC Struct. Biol. 10, 3), our pre-steady state data show that loss of single cationic residues can alter the rates of all DNA-linked steps in the reaction, as well as movement of PCNA on DNA. These results explain an earlier finding that individual arginines and lysines inside human PCNA are essential for polymerase δ processivity (Fukuda, K., Morioka, H., Imajou, S., Ikeda, S., Ohtsuka, E., and Tsurimoto, T. (1995) Structure-function relationship of the eukaryotic DNA replication factor, proliferating cell nuclear antigen. J. Biol. Chem. 270, 22527–22534). Mutations in the N-terminal domain have greater impact than in the C-terminal domain, indicating a positional bias in PCNA-DNA contacts that can influence its functions on DNA.

scholarly journals Cryo-EM structures reveal high-resolution mechanism of a DNA polymerase sliding clamp loader

2021 ◽  
Author(s):  
Christl Gaubitz ◽  
Xingchen Liu ◽  
Joshua Pajak ◽  
Nicholas P. Stone ◽  
Janelle A. Hayes ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Conformational Changes ◽  
Large Scale ◽  
Atp Hydrolysis ◽  
Nuclear Antigen ◽  
Clamp Loader ◽  
Aaa Atpase ◽  
Sliding Clamp ◽  
Sliding Clamps ◽  
Factor C

Sliding clamps are ring-shaped protein complexes that are integral to the DNA replication machinery of all life. Sliding clamps are opened and installed onto DNA by clamp loader AAA+ ATPase complexes. However, how a clamp loader opens and closes the sliding clamp around DNA is still unknown. Here, we describe structures of the S. cerevisiae clamp loader Replication Factor C (RFC) bound to its cognate sliding clamp Proliferating Cell Nuclear Antigen (PCNA) en route to successful loading. RFC first binds to PCNA in a dynamic, closed conformation that blocks both ATPase activity and DNA binding. RFC then opens the PCNA ring through a large-scale 'crab-claw' expansion of both RFC and PCNA that explains how RFC prefers initial binding of PCNA over DNA. Next, the open RFC:PCNA complex binds DNA and interrogates the primer-template junction using a surprising base-flipping mechanism. Our structures indicate that initial PCNA opening and subsequent closure around DNA do not require ATP hydrolysis, but are driven by binding energy. ATP hydrolysis, which is necessary for RFC release, is triggered by interactions with both PCNA and DNA, explaining RFC's switch-like ATPase activity. Our work reveals how a AAA+ machine undergoes dramatic conformational changes for achieving binding preference and substrate remodeling.

scholarly journals Structure of the human clamp loader bound to the sliding clamp: a further twist on AAA+ mechanism

2020 ◽  
Author(s):  
Christl Gaubitz ◽  
Xingchen Liu ◽  
Joseph Magrino ◽  
Nicholas P. Stone ◽  
Jacob Landeck ◽  
...  
Keyword(s):  
Active Sites ◽  
Mechanistic Explanation ◽  
Clamp Loader ◽  
Sliding Clamp ◽  
Disease Mutations ◽  
Clamp Loading ◽  
Clamp Loaders ◽  
Factor C ◽  
Aaa Atpases

SUMMARYDNA replication requires the sliding clamp, a ring-shaped protein complex that encircles DNA, where it acts as an essential cofactor for DNA polymerases and other proteins. The sliding clamp needs to be actively opened and installed onto DNA by a clamp loader ATPase of the AAA+ family. The human clamp loader Replication Factor C (RFC) and sliding clamp PCNA are both essential and play critical roles in several diseases. Despite decades of study, no structure of human RFC has been resolved. Here, we report the structure of human RFC bound to PCNA by cryo-EM to an overall resolution of ~3.4 Å. The active sites of RFC are fully bound to ATP analogs, which is expected to induce opening of the sliding clamp. However, we observe the complex in a conformation prior to PCNA opening, with the clamp loader ATPase modules forming an over-twisted spiral that is incapable of binding DNA or hydrolyzing ATP. The autoinhibited conformation observed here has many similarities to a previous yeast RFC:PCNA crystal structure, suggesting that eukaryotic clamp loaders adopt a similar autoinhibited state early on in clamp loading. Our results point to a ‘Limited Change/Induced Fit’ mechanism in which the clamp first opens, followed by DNA binding inducing opening of the loader to release auto-inhibition. The proposed change from an over-twisted to an active conformation reveals a novel regulatory mechanism for AAA+ ATPases. Finally, our structural analysis of disease mutations leads to a mechanistic explanation for the role of RFC in human health.

Sign in / Sign up

Export Citation Format

Share Document