scholarly journals The interplay of DNA binding, ATP hydrolysis and helicase activities of the archaeal MCM helicase

2011 ◽  
Vol 436 (2) ◽  
pp. 409-414 ◽  
Author(s):  
Li Phing Liew ◽  
Stephen D. Bell
Keyword(s):  
Dna Binding ◽  
Active Site ◽  
Atp Hydrolysis ◽  
Sulfolobus Solfataricus ◽  
Active Form ◽  
Dna Helicase ◽  
Atpase Domain ◽  
Minichromosome Maintenance ◽  
Conserved Residues ◽  
Mcm Helicase

The MCM (minichromosome maintenance) proteins of archaea are widely believed to be the replicative DNA helicase of these organisms. Most archaea possess a single MCM orthologue that forms homo-multimeric assemblies with a single hexamer believed to be the active form. In the present study we characterize the roles of highly conserved residues in the ATPase domain of the MCM of the hyperthermophilic archaeon Sulfolobus solfataricus. Our results identify a potential conduit for communicating DNA-binding information to the ATPase active site.

scholarly journals DNA Binding by the Methanothermobacter thermautotrophicus Cdc6 Protein Is Inhibited by the Minichromosome Maintenance Helicase

2006 ◽  
Vol 188 (12) ◽  
pp. 4577-4580 ◽  
Author(s):  
Rajesh Kasiviswanathan ◽  
Jae-Ho Shin ◽  
Zvi Kelman
Keyword(s):  
Dna Binding ◽  
Binding Activity ◽  
Minichromosome Maintenance ◽  
Single Stranded Dna ◽  
Mutant Proteins ◽  
Dna Binding Activity ◽  
Double Stranded Dna ◽  
Mcm Helicase ◽  
Methanothermobacter Thermautotrophicus

ABSTRACT The Cdc6 proteins from the archaeon Methanothermobacter thermautotrophicus were previously shown to bind double-stranded DNA. It is shown here that the proteins also bind single-stranded DNA. Using minichromosome maintenance (MCM) helicase mutant proteins unable to bind DNA, it was found that the interaction of MCM with Cdc6 inhibits the DNA binding activity of Cdc6.

scholarly journals Mutations in Subdomain B of the Minichromosome Maintenance (MCM) Helicase Affect DNA Binding and Modulate Conformational Transitions

2008 ◽  
Vol 284 (9) ◽  
pp. 5654-5661 ◽  
Author(s):  
Elizabeth R. Jenkinson ◽  
Alessandro Costa ◽  
Andrew P. Leech ◽  
Ardan Patwardhan ◽  
Silvia Onesti ◽  
...  
Keyword(s):  
Dna Binding ◽  
Conformational Transitions ◽  
Minichromosome Maintenance ◽  
Mcm Helicase

scholarly journals The large terminase DNA packaging motor grips DNA with its ATPase domain for cleavage by the flexible nuclease domain

2016 ◽  
Author(s):  
Brendan J. Hilbert ◽  
Janelle A. Hayes ◽  
Nicholas P. Stone ◽  
Rui-Gang Xu ◽  
Brian A. Kelch
Keyword(s):  
Dna Binding ◽  
Active Site ◽  
Dna Cleavage ◽  
Atpase Domain ◽  
Dna Translocation ◽  
Genome Packaging ◽  
Virus Maturation ◽  
Nuclease Domain ◽  
Dna Packaging Motor ◽  
Large Terminase

AbstractMany viruses use a powerful terminase motor to pump their genome inside an empty procapsid shell during virus maturation. The large terminase (TerL) protein contains both enzymatic activities necessary for packaging in such viruses: the ATPase that powers DNA translocation and an endonuclease that cleaves the concatemeric genome both at initiation and completion of genome packaging. However, how TerL binds DNA during translocation and cleavage is still mysterious. Here we investigate DNA binding and cleavage using TerL from the thermophilic phage P74-26. We report the structure of the P74-26 TerL nuclease domain, which allows us to model DNA binding in the nuclease active site. We screened a large panel of TerL variants for defects in binding and DNA cleavage, revealing that the ATPase domain is the primary site for DNA binding, and is required for nucleolysis. The nuclease domain is dispensable for DNA binding but residues lining the active site guide DNA for cleavage. Kinetic analysis of nucleolysis suggests flexible tethering of the nuclease domains during DNA cleavage. We propose that interactions with the procapsid shell during DNA translocation conformationally restrict the nuclease domain, inhibiting cleavage; TerL release from the procapsid upon completion of packaging unlocks the nuclease domains to cleave DNA.

scholarly journals The Sulfolobus solfataricus GINS Complex Stimulates DNA Binding and Processive DNA Unwinding by Minichromosome Maintenance Helicase

2015 ◽  
Vol 197 (21) ◽  
pp. 3409-3420 ◽  
Author(s):  
Shiwei Lang ◽  
Li Huang
Keyword(s):  
Dna Binding ◽  
Atpase Activity ◽  
Replication Fork ◽  
Molecular Motor ◽  
Sulfolobus Solfataricus ◽  
Dna Unwinding ◽  
Helicase Activity ◽  
Minichromosome Maintenance ◽  
Content Type ◽  
Double Stranded Dna

ABSTRACTGINS is a key component of the eukaryotic Cdc45-minichromosome maintenance (MCM)-GINS (CMG) complex, which unwinds duplex DNA at the moving replication fork. Archaeal GINS complexes have been shown to stimulate the helicase activity of their cognate MCM mainly by elevating its ATPase activity. Here, we report that GINS from the thermoacidophilic crenarchaeonSulfolobus solfataricus(SsoGINS) is capable of DNA binding and binds preferentially to single-stranded DNA (ssDNA) over double-stranded DNA (dsDNA). Notably, SsoGINS binds more strongly to dsDNA with a 5′ ssDNA tail than to dsDNA with a 3′ tail and more strongly to an ssDNA fragment blocked at the 3′ end than to one at the 5′ end with a biotin-streptavidin (SA) complex, suggesting the ability of the protein complex to slide in a 5′-to-3′ direction along ssDNA. DNA-bound SsoGINS enhances DNA binding by SsoMCM. Furthermore, SsoGINS increases the helicase activity of SsoMCM. However, the ATPase activity of SsoMCM is not affected by SsoGINS. Our results suggest that SsoGINS facilitates processive DNA unwinding by SsoMCM by enhancing the binding of the helicase to DNA. We propose that SsoGINS stabilizes the interaction of SsoMCM with the replication fork and moves along with the helicase as the fork progresses.IMPORTANCEGINS is a key component of the eukaryotic Cdc45-MCM-GINS complex, a molecular motor that drives the unwinding of DNA in front of the replication fork.Archaeaalso encode GINS, which interacts with MCM, the helicase. But how archaeal GINS serves its role remains to be understood. In this study, we show that GINS from the hyperthermophilic archaeonSulfolobus solfataricusis able to bind to DNA and slide along ssDNA in a 5′-to-3′ direction. Furthermore,SulfolobusGINS enhances DNA binding by MCM, which slides along ssDNA in a 3′-to-5′ direction. Taken together, these results suggest thatSulfolobusGINS may stabilize the interaction of MCM with the moving replication fork, facilitating processive DNA unwinding.

scholarly journals Analysis of the crystal structure of an active MCM hexamer

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Justin M Miller ◽  
Buenafe T Arachea ◽  
Leslie B Epling ◽  
Eric J Enemark
Keyword(s):  
Crystal Structure ◽  
Atp Hydrolysis ◽  
Pyrococcus Furiosus ◽  
Binding Pocket ◽  
Activation Mechanism ◽  
Binding Motif ◽  
Atpase Domain ◽  
Ssdna Binding ◽  
Allosteric Communication ◽  
Mcm Helicase

In a previous Research article (<xref ref-type="bibr" rid="bib25">Froelich et al., 2014</xref>), we suggested an MCM helicase activation mechanism, but were limited in discussing the ATPase domain because it was absent from the crystal structure. Here we present the crystal structure of a nearly full-length MCM hexamer that is helicase-active and thus has all features essential for unwinding DNA. The structure is a chimera of Sulfolobus solfataricus N-terminal domain and Pyrococcus furiosus ATPase domain. We discuss three major findings: 1) a novel conformation for the A-subdomain that could play a role in MCM regulation; 2) interaction of a universally conserved glutamine in the N-terminal Allosteric Communication Loop with the AAA+ domain helix-2-insert (h2i); and 3) a recessed binding pocket for the MCM ssDNA-binding motif influenced by the h2i. We suggest that during helicase activation, the h2i clamps down on the leading strand to facilitate strand retention and regulate ATP hydrolysis.

scholarly journals Differences in the Single-stranded DNA Binding Activities of MCM2-7 and MCM467

2007 ◽  
Vol 282 (46) ◽  
pp. 33795-33804 ◽  
Author(s):  
Matthew L. Bochman ◽  
Anthony Schwacha
Keyword(s):  
Dna Binding ◽  
Active Site ◽  
Biochemical Properties ◽  
Dna Helicase ◽  
Binding Activity ◽  
Helicase Activity ◽  
Single Stranded Dna ◽  
Ssdna Binding ◽  
Better Than

The MCM2-7 complex, a hexamer containing six distinct and essential subunits, is postulated to be the eukaryotic replicative DNA helicase. Although all six subunits function at the replication fork, only a specific subcomplex consisting of the MCM4, 6, and 7 subunits (MCM467) and not the MCM2-7 complex exhibits DNA helicase activity in vitro. To understand why MCM2-7 lacks helicase activity and to address the possible function of the MCM2, 3, and 5 subunits, we have compared the biochemical properties of the Saccharomyces cerevisiae MCM2-7 and MCM467 complexes. We demonstrate that both complexes are toroidal and possess a similar ATP-dependent single-stranded DNA (ssDNA) binding activity, indicating that the lack of helicase activity by MCM2-7 is not due to ineffective ssDNA binding. We identify two important differences between them. MCM467 binds dsDNA better than MCM2-7. In addition, we find that the rate of MCM2-7/ssDNA association is slow compared with MCM467; the association rate can be dramatically increased either by preincubation with ATP or by inclusion of mutations that ablate the MCM2/5 active site. We propose that the DNA binding differences between MCM2-7 and MCM467 correspond to a conformational change at the MCM2/5 active site with putative regulatory significance.

ATP Hydrolysis and DNA Binding Confer Thermostability on the MCM Helicase†

Biochemistry ◽  
2009 ◽  
Vol 48 (11) ◽  
pp. 2330-2339 ◽  
Author(s):  
Nozomi Sakakibara ◽  
Frederick P. Schwarz ◽  
Zvi Kelman
Keyword(s):  
Dna Binding ◽  
Atp Hydrolysis ◽  
Mcm Helicase

Structures of c-di-GMP/cGAMP degrading phosphodiesterase VcEAL: identification of a novel conformational switch and its implication

2019 ◽  
Vol 476 (21) ◽  
pp. 3333-3353 ◽  
Author(s):  
Malti Yadav ◽  
Kamalendu Pal ◽  
Udayaditya Sen
Keyword(s):  
Active Site ◽  
Metal Binding ◽  
Metal Ion ◽  
Triosephosphate Isomerase ◽  
Catalytic Mechanism ◽  
Degradation Mechanism ◽  
Structural Basis ◽  
Conserved Residues ◽  
Phosphodiester Bond ◽  
Cyclic Dinucleotides

Cyclic dinucleotides (CDNs) have emerged as the central molecules that aid bacteria to adapt and thrive in changing environmental conditions. Therefore, tight regulation of intracellular CDN concentration by counteracting the action of dinucleotide cyclases and phosphodiesterases (PDEs) is critical. Here, we demonstrate that a putative stand-alone EAL domain PDE from Vibrio cholerae (VcEAL) is capable to degrade both the second messenger c-di-GMP and hybrid 3′3′-cyclic GMP–AMP (cGAMP). To unveil their degradation mechanism, we have determined high-resolution crystal structures of VcEAL with Ca2+, c-di-GMP-Ca2+, 5′-pGpG-Ca2+ and cGAMP-Ca2+, the latter provides the first structural basis of cGAMP hydrolysis. Structural studies reveal a typical triosephosphate isomerase barrel-fold with substrate c-di-GMP/cGAMP bound in an extended conformation. Highly conserved residues specifically bind the guanine base of c-di-GMP/cGAMP in the G2 site while the semi-conserved nature of residues at the G1 site could act as a specificity determinant. Two metal ions, co-ordinated with six stubbornly conserved residues and two non-bridging scissile phosphate oxygens of c-di-GMP/cGAMP, activate a water molecule for an in-line attack on the phosphodiester bond, supporting two-metal ion-based catalytic mechanism. PDE activity and biofilm assays of several prudently designed mutants collectively demonstrate that VcEAL active site is charge and size optimized. Intriguingly, in VcEAL-5′-pGpG-Ca2+ structure, β5–α5 loop adopts a novel conformation that along with conserved E131 creates a new metal-binding site. This novel conformation along with several subtle changes in the active site designate VcEAL-5′-pGpG-Ca2+ structure quite different from other 5′-pGpG bound structures reported earlier.

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