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.

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 Mechanisms of loading and release of the 9-1-1 checkpoint clamp

2021 ◽  
Author(s):  
Juan C Castaneda ◽  
Marina Schrecker ◽  
Dirk Remus ◽  
Richard K Hite
Keyword(s):  
Conformational Changes ◽  
Atp Hydrolysis ◽  
Clamp Loader ◽  
Opposite Orientation ◽  
Checkpoint Kinase ◽  
Sliding Clamp ◽  
Double Stranded Dna ◽  
Clamp Loading ◽  
Clamp Loaders ◽  
Open States

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.

scholarly journals Allosteric communication in DNA polymerase clamp loaders relies on a critical hydrogen-bonded junction

2021 ◽  
Author(s):  
Subu Subramanian ◽  
Kent Gorday ◽  
Kendra Marcus ◽  
Matthew R. Orellana ◽  
Peter Ren ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Clamp Loader ◽  
Sliding Clamp ◽  
Allosteric Communication ◽  
Clamp Loaders ◽  
T4 Bacteriophage ◽  
Network Of Hydrogen Bonds ◽  
Dynamics Simulations ◽  
Aaa Atpases ◽  
Hydrogen Bonded

ABSTRACTClamp loaders are AAA+ ATPases that load sliding clamps onto DNA. We mapped the mutational sensitivity of the T4 bacteriophage sliding clamp and clamp loader by deep mutagenesis, and found that residues not involved in catalysis or binding display remarkable tolerance to mutation. An exception is a glutamine residue in the AAA+ module (Gln 118) that is not located at a catalytic or interfacial site. Gln 118 forms a hydrogen-bonded junction in a helical unit that we term the central coupler, because it connects the catalytic centers to DNA and the sliding clamp. A suppressor mutation indicates that hydrogen bonding in the junction is important, and molecular dynamics simulations reveal that it maintains rigidity in the central coupler. The glutamine-mediated junction is preserved in diverse AAA+ ATPases, suggesting that a connected network of hydrogen bonds that links ATP molecules is an essential aspect of allosteric communication in these proteins.

scholarly journals Allosteric communication in DNA polymerase clamp loaders relies on a critical hydrogen-bonded junction

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Subu Subramanian ◽  
Kent Gorday ◽  
Kendra Marcus ◽  
Matthew R Orellana ◽  
Peter Ren ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Clamp Loader ◽  
Sliding Clamp ◽  
Allosteric Communication ◽  
Clamp Loaders ◽  
T4 Bacteriophage ◽  
Network Of Hydrogen Bonds ◽  
Dynamics Simulations ◽  
Aaa Atpases ◽  
Hydrogen Bonded

Clamp loaders are AAA+ ATPases that load sliding clamps onto DNA. We mapped the mutational sensitivity of the T4 bacteriophage sliding clamp and clamp loader by deep mutagenesis, and found that residues not involved in catalysis or binding display remarkable tolerance to mutation. An exception is a glutamine residue in the AAA+ module (Gln 118) that is not located at a catalytic or interfacial site. Gln 118 forms a hydrogen-bonded junction in a helical unit that we term the central coupler, because it connects the catalytic centers to DNA and the sliding clamp. A suppressor mutation indicates that hydrogen bonding in the junction is important, and molecular dynamics simulations reveal that it maintains rigidity in the central coupler. The glutamine-mediated junction is preserved in diverse AAA+ ATPases, suggesting that a connected network of hydrogen bonds that links ATP molecules is an essential aspect of allosteric communication in these proteins.

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 Eukaryotic clamp loaders and unloaders in the maintenance of genome stability

2020 ◽  
Vol 52 (12) ◽  
pp. 1948-1958
Author(s):  
Kyoo-young Lee ◽  
Su Hyung Park
Keyword(s):  
Dna Damage ◽  
Genomic Instability ◽  
Genome Stability ◽  
Critical Role ◽  
Nuclear Antigen ◽  
Checkpoint Activation ◽  
Sliding Clamp ◽  
Clamp Loaders ◽  
Factor C

AbstractEukaryotic sliding clamp proliferating cell nuclear antigen (PCNA) plays a critical role as a processivity factor for DNA polymerases and as a binding and acting platform for many proteins. The ring-shaped PCNA homotrimer and the DNA damage checkpoint clamp 9-1-1 are loaded onto DNA by clamp loaders. PCNA can be loaded by the pentameric replication factor C (RFC) complex and the CTF18-RFC-like complex (RLC) in vitro. In cells, each complex loads PCNA for different purposes; RFC-loaded PCNA is essential for DNA replication, while CTF18-RLC-loaded PCNA participates in cohesion establishment and checkpoint activation. After completing its tasks, PCNA is unloaded by ATAD5 (Elg1 in yeast)-RLC. The 9-1-1 clamp is loaded at DNA damage sites by RAD17 (Rad24 in yeast)-RLC. All five RFC complex components, but none of the three large subunits of RLC, CTF18, ATAD5, or RAD17, are essential for cell survival; however, deficiency of the three RLC proteins leads to genomic instability. In this review, we describe recent findings that contribute to the understanding of the basic roles of the RFC complex and RLCs and how genomic instability due to deficiency of the three RLCs is linked to the molecular and cellular activity of RLC, particularly focusing on ATAD5 (Elg1).

scholarly journals Stepwise assembly of the human replicative polymerase holoenzyme

eLife ◽  
2013 ◽  
Vol 2 ◽  
Author(s):  
Mark Hedglin ◽  
Senthil K Perumal ◽  
Zhenxin Hu ◽  
Stephen Benkovic
Keyword(s):  
Dna Polymerase ◽  
Clamp Loader ◽  
Dna Interactions ◽  
Sliding Clamp ◽  
Stepwise Manner ◽  
Protein Dna Interactions ◽  
Clamp Loaders ◽  
Sliding Clamps ◽  
Stepwise Assembly

In most organisms, clamp loaders catalyze both the loading of sliding clamps onto DNA and their removal. How these opposing activities are regulated during assembly of the DNA polymerase holoenzyme remains unknown. By utilizing FRET to monitor protein-DNA interactions, we examined assembly of the human holoenzyme. The results indicate that assembly proceeds in a stepwise manner. The clamp loader (RFC) loads a sliding clamp (PCNA) onto a primer/template junction but remains transiently bound to the DNA. Unable to slide away, PCNA re-engages with RFC and is unloaded. In the presence of polymerase (polδ), loaded PCNA is captured from DNA-bound RFC which subsequently dissociates, leaving behind the holoenzyme. These studies suggest that the unloading activity of RFC maximizes the utilization of PCNA by inhibiting the build-up of free PCNA on DNA in the absence of polymerase and recycling limited PCNA to keep up with ongoing replication.

scholarly journals Molecular Analyses of a Three-Subunit Euryarchaeal Clamp Loader Complex from Methanosarcina acetivorans

2009 ◽  
Vol 191 (21) ◽  
pp. 6539-6549 ◽  
Author(s):  
Yi-Hsing Chen ◽  
Yuyen Lin ◽  
Aya Yoshinaga ◽  
Benazir Chhotani ◽  
Jenna L. Lorenzini ◽  
...  
Keyword(s):  
Spatial Distribution ◽  
Dna Synthesis ◽  
Mutational Analysis ◽  
Large Subunit ◽  
Size Exclusion ◽  
Clamp Loader ◽  
Methanosarcina Acetivorans ◽  
Chromosomal Dna ◽  
Clamp Loaders ◽  
Factor C

ABSTRACT Chromosomal DNA replication is dependent on processive DNA synthesis. Across the three domains of life and in certain viruses, a toroidal sliding clamp confers processivity to replicative DNA polymerases by encircling the DNA and engaging the polymerase in protein/protein interactions. Sliding clamps are ring-shaped; therefore, they have cognate clamp loaders that open and load them onto DNA. Here we use biochemical and mutational analyses to study the structure/function of the Methanosarcina acetivorans clamp loader or replication factor C (RFC) homolog. M. acetivorans RFC (RFC Ma ), which represents an intermediate between the common archaeal RFC and the eukaryotic RFC, comprises two different small subunits (RFCS1 and RFCS2) and a large subunit (RFCL). Size exclusion chromatography suggested that RFCS1 exists in oligomeric states depending on protein concentration, while RFCS2 exists as a monomer. Protein complexes of RFCS1/RFCS2 formed in solution; however, they failed to stimulate DNA synthesis by a cognate DNA polymerase in the presence of its clamp. Determination of the subunit composition and previous mutational analysis allowed the prediction of the spatial distribution of subunits in this new member of the clamp loader family. Three RFCS1 subunits are flanked by an RFCS2 and an RFCL. The spatial distribution is, therefore, reminiscent of the minimal Escherichia coli clamp loader that exists in space as three γ-subunits (motor) flanked by the δ′ (stator) and the δ (wrench) subunits. Mutational analysis, however, suggested that the similarity between the two clamp loaders does not translate into the complete conservation of the functions of individual subunits within the RFC Ma complex.

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.

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