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

2001 ◽  
Vol 6 (8) ◽  
pp. 699-706 ◽  
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

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 How a DNA Polymerase Clamp Loader Opens a Sliding Clamp

Science ◽  
2011 ◽  
Vol 334 (6063) ◽  
pp. 1675-1680 ◽  
Author(s):  
B. A. Kelch ◽  
D. L. Makino ◽  
M. O'Donnell ◽  
J. Kuriyan
Keyword(s):  
Dna Polymerase ◽  
Clamp Loader ◽  
Sliding Clamp

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

eLife ◽  
2015 ◽  
Vol 4 ◽  
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.

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

2017 ◽  
Vol 45 (8) ◽  
pp. 4550-4563 ◽  
Author(s):  
Ryo Fujisawa ◽  
Eiji Ohashi ◽  
Kouji Hirota ◽  
Toshiki Tsurimoto
Keyword(s):  
Dna Polymerase ◽  
Clamp Loader ◽  
Sliding Clamp

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.

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