How a DNA Polymerase Clamp Loader Opens a Sliding Clamp

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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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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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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Human CTF18-RFC clamp-loader complexed with non-synthesising DNA polymerase ε efficiently loads the PCNA sliding clamp

Nucleic Acids Research ◽

10.1093/nar/gkx096 ◽

2017 ◽

Vol 45 (8) ◽

pp. 4550-4563 ◽

Cited By ~ 11

Author(s):

Ryo Fujisawa ◽

Eiji Ohashi ◽

Kouji Hirota ◽

Toshiki Tsurimoto

Keyword(s):

Dna Polymerase ◽

Clamp Loader ◽

Sliding Clamp

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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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Functional interactions of an archaeal sliding clamp with mammalian clamp loader and DNA polymerase δ

Genes to Cells ◽

10.1046/j.1365-2443.2001.00451.x ◽

2001 ◽

Vol 6 (8) ◽

pp. 699-706 ◽

Cited By ~ 20

Author(s):

Yoshizumi Ishino ◽

Toshiki Tsurimoto ◽

Sonoko Ishino ◽

Isaac K. O. Cann

Keyword(s):

Dna Polymerase ◽

Clamp Loader ◽

Dna Polymerase Δ ◽

Sliding Clamp ◽

Functional Interactions

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

eLife ◽

10.7554/elife.66181 ◽

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.

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Faculty Opinions recommendation of Mechanism of processivity clamp opening by the delta subunit wrench of the clamp loader complex of E. coli DNA polymerase III.

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

10.3410/f.1001104.10052 ◽

2001 ◽

Author(s):

Janet Lindsley

Keyword(s):

Dna Polymerase ◽

Dna Polymerase Iii ◽

Clamp Loader ◽

E Coli ◽

Polymerase Iii ◽

Delta Subunit

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The SOS Error-Prone DNA Polymerase V Mutasome and β-Sliding Clamp Acting in Concert on Undamaged DNA and during Translesion Synthesis

Cells ◽

10.3390/cells10051083 ◽

2021 ◽

Vol 10 (5) ◽

pp. 1083

Author(s):

Adhirath Sikand ◽

Malgorzata Jaszczur ◽

Linda B. Bloom ◽

Roger Woodgate ◽

Michael M. Cox ◽

...

Keyword(s):

Dna Synthesis ◽

Dna Polymerase ◽

Pathogenic Bacteria ◽

Fold Increase ◽

Translesion Dna Synthesis ◽

Sliding Clamp ◽

Pol V ◽

Dna Polymerase V ◽

Acting In Concert

In the mid 1970s, Miroslav Radman and Evelyn Witkin proposed that Escherichia coli must encode a specialized error-prone DNA polymerase (pol) to account for the 100-fold increase in mutations accompanying induction of the SOS regulon. By the late 1980s, genetic studies showed that SOS mutagenesis required the presence of two “UV mutagenesis” genes, umuC and umuD, along with recA. Guided by the genetics, decades of biochemical studies have defined the predicted error-prone DNA polymerase as an activated complex of these three gene products, assembled as a mutasome, pol V Mut = UmuD’2C-RecA-ATP. Here, we explore the role of the β-sliding processivity clamp on the efficiency of pol V Mut-catalyzed DNA synthesis on undamaged DNA and during translesion DNA synthesis (TLS). Primer elongation efficiencies and TLS were strongly enhanced in the presence of β. The results suggest that β may have two stabilizing roles: its canonical role in tethering the pol at a primer-3’-terminus, and a possible second role in inhibiting pol V Mut’s ATPase to reduce the rate of mutasome-DNA dissociation. The identification of umuC, umuD, and recA homologs in numerous strains of pathogenic bacteria and plasmids will ensure the long and productive continuation of the genetic and biochemical journey initiated by Radman and Witkin.

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