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

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 ClpX is Essential and Activated by Single Strand DNA Binding Protein in Mycobacteria

2020 ◽  
Author(s):  
Jemila C. Kester ◽  
Olga Kandror ◽  
Tatos Akopian ◽  
Michael R. Chase ◽  
Junhao Zhu ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Dna Replication ◽  
Dna Binding ◽  
Drug Targets ◽  
Atp Hydrolysis ◽  
Binding Protein ◽  
Dna Binding Protein ◽  
Cellular Functions ◽  
Mycobacterial Growth ◽  
Dna Maintenance

The ClpP1P2 proteolytic complex is essential in Mycobacterium tuberculosis (Mtb). Proteolysis by ClpP1P2 requires an associated ATPase, either ClpX or ClpC1. Here, we seek to define the unique contributions of the ClpX ATPase to mycobacterial growth. We formally demonstrate that ClpX is essential for mycobacterial growth and to understand its essential functions, we identify ClpX-His-interacting proteins by pulldown and tandem mass spectrometry. We find an unexpected association between ClpX and proteins involved in DNA replication, and confirm a physical association between ClpX and the essential DNA maintenance protein Single-Stranded DNA Binding protein (SSB). Purified SSB is not degraded by ClpXP1P2; instead SSB enhances ATP hydrolysis by ClpX and degradation of the model substrate GFP-SsrA by ClpXP1P2. This activation of ClpX is mediated by the C-terminal tail of SSB that had been implicated in the activation of other ATPases associated with DNA replication. Consistent with the predicted interactions, depletion of clpX transcript perturbs DNA replication. These data reveal that ClpX participates in DNA replication and identify the first activator of ClpX in mycobacteria. IMPORTANCE Tuberculosis, caused by Mycobacterium tuberculosis, imposes a major global health burden, surpassing HIV and malaria in annual deaths. The ClpP1P2 proteolytic complex and its cofactor ClpX are attractive drug targets, but their precise cellular functions are unclear. This work confirms ClpX’s essentiality and describes a novel interaction between ClpX and SSB, a component of the DNA replication machinery. Further, we demonstrate that a loss of ClpX is sufficient to interrupt DNA replication, suggesting the ClpX-SSB complex may play a role in DNA replication in mycobacteria.

scholarly journals An ATP/ADP-Dependent Molecular Switch Regulates the Stability of p53-DNA Complexes

1999 ◽  
Vol 19 (11) ◽  
pp. 7501-7510 ◽  
Author(s):  
Andrei L. Okorokov ◽  
Jo Milner
Keyword(s):  
Dna Damage ◽  
Dna Binding ◽  
Cellular Response ◽  
Atp Hydrolysis ◽  
P53 Protein ◽  
Molecular Switch ◽  
Dna Complexes ◽  
Switch Mechanism ◽  
The Stability ◽  
First Time

ABSTRACT Interaction with DNA is essential for the tumor suppressor functions of p53. We now show, for the first time, that the interaction of p53 with DNA can be stabilized by small molecules, such as ADP and dADP. Our results also indicate an ATP/ADP molecular switch mechanism which determines the off-on states for p53-DNA binding. This ATP/ADP molecular switch requires dimer-dimer interaction of the p53 tetramer. Dissociation of p53-DNA complexes by ATP is independent of ATP hydrolysis. Low-level ATPase activity is nonetheless associated with ATP-p53 interaction and may serve to regenerate ADP-p53, thus recycling the high-affinity DNA binding form of p53. The ATP/ADP regulatory mechanism applies to two distinct types of p53 interaction with DNA, namely, sequence-specific DNA binding (via the core domain of the p53 protein) and binding to sites of DNA damage (via the C-terminal domain). Further studies indicate that ADP not only stabilizes p53-DNA complexes but also renders the complexes susceptible to dissociation by specific p53 binding proteins. We propose a model in which the DNA binding functions of p53 are regulated by an ATP/ADP molecular switch, and we suggest that this mechanism may function during the cellular response to DNA damage.

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

Archaeal Hel308 Domain V Couples DNA Binding to ATP Hydrolysis and Positions DNA for Unwinding Over the Helicase Ratchet

2007 ◽  
Vol 374 (5) ◽  
pp. 1139-1144 ◽  
Author(s):  
Isabel L. Woodman ◽  
Geoffrey S. Briggs ◽  
Edward L. Bolt
Keyword(s):  
Dna Binding ◽  
Atp Hydrolysis ◽  
Domain V

scholarly journals Cdc6 ATPase activity disengages Cdc6 from the pre-replicative complex to promote DNA replication

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
FuJung Chang ◽  
Alberto Riera ◽  
Cecile Evrin ◽  
Jingchuan Sun ◽  
Huilin Li ◽  
...  
Keyword(s):  
Dna Replication ◽  
Atpase Activity ◽  
Atp Hydrolysis ◽  
S Phase ◽  
G1 Phase ◽  
Helicase Loading ◽  
Mcm Helicase

To initiate DNA replication, cells first load an MCM helicase double hexamer at origins in a reaction requiring ORC, Cdc6, and Cdt1, also called pre-replicative complex (pre-RC) assembly. The essential mechanistic role of Cdc6 ATP hydrolysis in this reaction is still incompletely understood. Here, we show that although Cdc6 ATP hydrolysis is essential to initiate DNA replication, it is not essential for MCM loading. Using purified proteins, an ATPase-defective Cdc6 mutant ‘Cdc6-E224Q’ promoted MCM loading on DNA. Cdc6-E224Q also promoted MCM binding at origins in vivo but cells remained blocked in G1-phase. If after loading MCM, Cdc6-E224Q was degraded, cells entered an apparently normal S-phase and replicated DNA, a phenotype seen with two additional Cdc6 ATPase-defective mutants. Cdc6 ATP hydrolysis is therefore required for Cdc6 disengagement from the pre-RC after helicase loading to advance subsequent steps in helicase activation in vivo.

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.

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