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 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 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.

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.

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
Keyword(s):  
Dna Damage ◽  
Dna Binding ◽  
Atp Hydrolysis ◽  
Dna Damage Checkpoint ◽  
Clamp Loader ◽  
Dna Binding Site ◽  
Clamp Loading ◽  
Open Ring ◽  
Binding Residues ◽  
Higher Eukaryotes

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.

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.

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