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

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.

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

10.1101/2020.12.31.424921 ◽

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.

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Stepwise assembly of the human replicative polymerase holoenzyme

eLife ◽

10.7554/elife.00278 ◽

2013 ◽

Vol 2 ◽

Cited By ~ 20

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.

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

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2007437117 ◽

2020 ◽

Vol 117 (38) ◽

pp. 23571-23580 ◽

Cited By ~ 1

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.

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

10.1101/2020.02.18.953257 ◽

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.

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

10.1101/2021.09.13.460164 ◽

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.

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Faculty Opinions recommendation of How a DNA polymerase clamp loader opens a sliding clamp.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.13988985.15445098 ◽

2012 ◽

Author(s):

Tim Clausen ◽

Alexander Heuck

Keyword(s):

Dna Polymerase ◽

Clamp Loader ◽

Sliding Clamp

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How a DNA Polymerase Clamp Loader Opens a Sliding Clamp

Science ◽

10.1126/science.1211884 ◽

2011 ◽

Vol 334 (6063) ◽

pp. 1675-1680 ◽

Cited By ~ 110

Author(s):

B. A. Kelch ◽

D. L. Makino ◽

M. O'Donnell ◽

J. Kuriyan

Keyword(s):

Dna Polymerase ◽

Clamp Loader ◽

Sliding Clamp

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cryo-EM structures of the E. coli replicative DNA polymerase reveal its dynamic interactions with the DNA sliding clamp, exonuclease and τ

eLife ◽

10.7554/elife.11134 ◽

2015 ◽

Vol 4 ◽

Cited By ~ 40

Author(s):

Rafael Fernandez-Leiro ◽

Julian Conrad ◽

Sjors HW Scheres ◽

Meindert H Lamers

Keyword(s):

Dna Polymerase ◽

Bound State ◽

Tail Domain ◽

Clamp Loader ◽

Dynamic Interactions ◽

Catalytic Core ◽

E Coli ◽

Sliding Clamp ◽

Cryo Electron Microscopy ◽

Multiple Contacts

The replicative DNA polymerase PolIIIα from Escherichia coli is a uniquely fast and processive enzyme. For its activity it relies on the DNA sliding clamp β, the proofreading exonuclease ε and the C-terminal domain of the clamp loader subunit τ. Due to the dynamic nature of the four-protein complex it has long been refractory to structural characterization. Here we present the 8 Å resolution cryo-electron microscopy structures of DNA-bound and DNA-free states of the PolIII-clamp-exonuclease-τc complex. The structures show how the polymerase is tethered to the DNA through multiple contacts with the clamp and exonuclease. A novel contact between the polymerase and clamp is made in the DNA bound state, facilitated by a large movement of the polymerase tail domain and τc. These structures provide crucial insights into the organization of the catalytic core of the replisome and form an important step towards determining the structure of the complete holoenzyme.

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