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 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 Coupling of ATPase activity, microtubule binding and mechanics in the dynein motor domain

2018 ◽  
Author(s):  
Stefan Niekamp ◽  
Nicolas Coudray ◽  
Nan Zhang ◽  
Ronald D. Vale ◽  
Gira Bhabha
Keyword(s):  
Atpase Activity ◽  
Conformational Change ◽  
Conformational Changes ◽  
Large Scale ◽  
Atp Hydrolysis ◽  
Molecular Motor ◽  
Coiled Coil ◽  
Microtubule Binding ◽  
Dynein Motor ◽  
In The Wild

The movement of a molecular motor protein along a cytoskeletal track requires communication between enzymatic, polymer-binding, and mechanical elements. Such communication is particularly complex and not well understood in the dynein motor, an ATPase that is comprised of a ring of six AAA domains, a large mechanical element (linker) spanning over the ring, and a microtubule-binding domain (MTBD) that is separated from the AAA ring by a ~135 Å coiled-coil stalk. We identified mutations in the stalk that disrupt directional motion, have microtubule-independent hyperactive ATPase activity, and nucleotide-independent low affinity for microtubules. Cryo-electron microscopy structures of a mutant that uncouples ATPase activity from directional movement reveal that nucleotide-dependent conformational changes occur normally in one half of the AAA ring, but are disrupted in the other half. The large-scale linker conformational change observed in the wild-type protein is also inhibited, revealing that this conformational change is not required for ATP hydrolysis. These results demonstrate an essential role of the stalk in regulating motor activity and coupling conformational changes across the two halves of the AAA ring.

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 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 Conformational heterogeneity of the AAA ATPase p97 characterized by single particle cryo-EM

2014 ◽  
Vol 70 (a1) ◽  
pp. C853-C853
Author(s):  
Driss Mountassif ◽  
Lucien Fabre ◽  
Kaustuv Basu ◽  
Mihnea Bostina ◽  
Slavica Jonic ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Conformational Changes ◽  
Single Particle ◽  
Molecular Mechanisms ◽  
Protein Complexes ◽  
Normal Mode Analysis ◽  
Single Amino Acid ◽  
Mode Analysis ◽  
Conformational Heterogeneity ◽  
Aaa Atpase

p97, a member of the AAA (ATPase Associated with various Activities) ATPase family, is essential and centrally involved in a wide variety of cellular processes. Single amino-acid substitutions in p97 have been associated with the severe degenerative disorder of Inclusion Body Myopathy associated with Paget disease of bone and Frontotemporal Dementia (IBMPFD) as well as amytropic leteral sclerosis (ALS). Current models propose that p97 acts as a motor transmitting the energy from the ATPase cycle to conformational changes of substrate protein complexes causing segregation, remodeling or translocation. Mutations at the interface between the N and the D1 domains impact the ATPase activity and the conformation of D2 on the opposite side of the protein complex, suggesting intermolecular communication. Because of limited structural information, the molecular mechanisms on how p97 drives its activities and the molecular basis for transmission of information within the molecule remain elusive. Structural heterogeneity is observed in vitro and is likely relevant for the in vivo biological function of p97. Single particle cryo-EM is the method of choice to study a flexible complex. The technique allows study in solution and also deals with sample heterogeneity by image classification. We have set-up the characterization of the conformational heterogeneity in WT and disease relevant p97 mutant using multi-likelihood classification and Hybrid Electron Microscopy Normal Mode Analysis HEMNMA. The multi-likelihood analysis shows a link between the conformations of the N and D2 domains. HEMNMA allows the analysis of the asymmetry of the conformational changes. Together these studies describe the structural flexibility of p97 and the coupling of the ATPase activity with conformational changes in health and in disease. Study of this model system also allows the development of new methods to understand the conformational heterogeneity of other protein complexes.

scholarly journals Regulation of RelA (p65) Function by the Large Subunit of Replication Factor C

2003 ◽  
Vol 23 (2) ◽  
pp. 721-732 ◽  
Author(s):  
Lisa A. Anderson ◽  
Neil D. Perkins
Keyword(s):  
Tumor Suppressor ◽  
Large Subunit ◽  
Dominant Negative ◽  
Nuclear Antigen ◽  
Replication Factor C ◽  
Clamp Loader ◽  
Replication Factor ◽  
Factor C

ABSTRACT The RelA (p65) subunit of NF-κB is an important regulator of inflammation, proliferation, and apoptosis. We have discovered that the large subunit, p140, of replication factor C (RFC) can function as a regulator of RelA. RFC is a clamp loader, facilitating the addition and removal of proliferating-cell nuclear antigen from DNA during replication and repair but can also interact directly with the retinoblastoma tumor suppressor protein and the transcription factor C/EBPα. We find that RFC (p140) interacts with RelA both in vitro and in vivo and stimulates RelA transactivation. In contrast, coexpression of fragments of RFC (p140) that mediate the interaction with RelA results in transcriptional inhibition. The significance of this regulation was confirmed by using short interfering RNA oligonucleotides targeted to RFC (p140). Down regulation of endogenous RFC (p140) inhibits expression from a chromosomally integrated reporter plasmid induced by endogenous, TNF-α-activated NF-κB. Dominant negative fragments of RFC (p140) also cooperate with overexpressed RelA to induce cell death. Interestingly, RFC (p140) also interacts with the tumor suppressor p53. Taken together, these observations suggest that, in addition to its previously described function in DNA replication and repair, RFC (p140) has an important role as a regulator of transcription and NF-κB activity.

scholarly journals Cryo-EM structures reveal how ATP and DNA binding in MutS coordinate the sequential steps of DNA mismatch repair

2021 ◽  
Author(s):  
Alessandro Borsellini ◽  
Vladislav Kunetsky ◽  
Peter Friedhoff ◽  
Meindert H. Lamers
Keyword(s):  
Dna Binding ◽  
Mismatch Repair ◽  
Conformational Changes ◽  
Atp Hydrolysis ◽  
Dna Mismatch Repair ◽  
Atp Binding ◽  
Dna Mismatch ◽  
Sliding Clamp ◽  
Adp Release ◽  
Atp Binding And Hydrolysis

DNA mismatch repair detects and removes mismatches from DNA reducing the error rate of DNA replication a 100-1000 fold. The MutS protein is one of the key players that scans for mismatches and coordinates the repair cascade. During this, MutS undergoes multiple conformational changes that initiate the subsequent steps, in response to ATP binding, hydrolysis, and release. How ATP induces the different conformations in MutS is not well understood. Here we present four cryo-EM structures of Escherichia coli MutS at sequential stages of the ATP hydrolysis cycle. These structures reveal how ATP binding and hydrolysis induces a closing and opening of the MutS dimer, respectively. Additional biophysical analysis furthermore explains how DNA binding modulates the ATPase cycle by preventing hydrolysis during scanning and mismatch binding, while preventing ADP release in the sliding clamp state. Nucleotide release is achieved when MutS encounters single stranded DNA that is produced during the removal of the daughter strand. This way, the combination of the ATP binding and hydrolysis and its modulation by DNA enable MutS to adopt different conformations needed to coordinate the sequential steps of the mismatch repair cascade.

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.

Sign in / Sign up

Export Citation Format

Share Document