scholarly journals An aromatic-rich loop couples DNA binding and ATP hydrolysis in the PriA DNA helicase

2016 ◽  
pp. gkw690 ◽  
Author(s):  
Tricia A. Windgassen ◽  
James L. Keck
Keyword(s):  
Dna Binding ◽  
Atp Hydrolysis ◽  
Dna 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 Unwinding Is an MCM Complex-dependent and ATP Hydrolysis-dependent Process

2004 ◽  
Vol 279 (44) ◽  
pp. 45586-45593 ◽  
Author(s):  
David Shechter ◽  
Carol Y. Ying ◽  
Jean Gautier
Keyword(s):  
Dna Replication ◽  
Neutralizing Antibodies ◽  
Atp Hydrolysis ◽  
Initial Period ◽  
Dna Helicase ◽  
Dna Unwinding ◽  
Origin Firing ◽  
Replicative Helicase ◽  
Mcm Proteins

Minichromosome maintenance proteins (Mcm) are essential in all eukaryotes and are absolutely required for initiation of DNA replication. The eukaryotic and archaeal Mcm proteins have conserved helicase motifs and exhibit DNA helicase and ATP hydrolysis activitiesin vitro. Although the Mcm proteins have been proposed to be the replicative helicase, the enzyme that melts the DNA helix at the replication fork, their function during cellular DNA replication elongation is still unclear. Using nucleoplasmic extract (NPE) fromXenopus laeviseggs and six purified polyclonal antibodies generated against each of theXenopusMcm proteins, we have demonstrated that Mcm proteins are required during DNA replication and DNA unwinding after initiation of replication. Quantitative depletion of Mcms from the NPE results in normal replication and unwinding, confirming that Mcms are required before pre-replicative complex assembly and dispensable thereafter. Replication and unwinding are inhibited when pooled neutralizing antibodies against the six different Mcm2–7 proteins are added during NPE incubation. Furthermore, replication is blocked by the addition of the Mcm antibodies after an initial period of replication in the NPE, visualized by a pulse of radiolabeled nucleotide at the same time as antibody addition. Addition of the cyclin-dependent kinase 2 inhibitor p21cip1specifically blocks origin firing but does not prevent helicase action. When p21cip1is added, followed by the non-hydrolyzable analog ATPγS to block helicase function, unwinding is inhibited, demonstrating that plasmid unwinding is specifically attributable to an ATP hydrolysis-dependent function. These data support the hypothesis that the Mcm protein complex functions as the replicative helicase.

scholarly journals DNA and ATP Binding Activities of the Baculovirus DNA Helicase P143

2001 ◽  
Vol 75 (15) ◽  
pp. 7206-7209 ◽  
Author(s):  
Vivien V. McDougal ◽  
Linda A. Guarino
Keyword(s):  
Conformational Change ◽  
Atp Hydrolysis ◽  
Protein Complexes ◽  
Dna Helicase ◽  
Atp Binding ◽  
Dna Unwinding ◽  
Single Stranded Dna ◽  
Inchworm Mechanism

ABSTRACT P143 is a DNA helicase that tightly binds both double-stranded and single-stranded DNA. DNA-protein complexes rapidly dissociated in the presence of ATP and Mg2+. This finding suggests that ATP hydrolysis causes a conformational change in P143 which decreases affinity for DNA. This supports the model of an inchworm mechanism of DNA unwinding.

scholarly journals Fanconi anemia group J mutation abolishes its DNA repair function by uncoupling DNA translocation from helicase activity or disruption of protein-DNA complexes

Blood ◽  
2010 ◽  
Vol 116 (19) ◽  
pp. 3780-3791 ◽  
Author(s):  
Yuliang Wu ◽  
Joshua A. Sommers ◽  
Avvaru N. Suhasini ◽  
Thomas Leonard ◽  
Julianna S. Deakyne ◽  
...  
Keyword(s):  
Dna Repair ◽  
Fanconi Anemia ◽  
Atp Hydrolysis ◽  
Bone Marrow Failure ◽  
Dna Helicase ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Dna Complexes ◽  
Null Cell ◽  
Negative Effect

Abstract Fanconi anemia (FA) is a genetic disease characterized by congenital abnormalities, bone marrow failure, and susceptibility to leukemia and other cancers. FANCJ, one of 13 genes linked to FA, encodes a DNA helicase proposed to operate in homologous recombination repair and replicational stress response. The pathogenic FANCJ-A349P amino acid substitution resides immediately adjacent to a highly conserved cysteine of the iron-sulfur domain. Given the genetic linkage of the FANCJ-A349P allele to FA, we investigated the effect of this particular mutation on the biochemical and cellular functions of the FANCJ protein. Purified recombinant FANCJ-A349P protein had reduced iron and was defective in coupling adenosine triphosphate (ATP) hydrolysis and translocase activity to unwinding forked duplex or G-quadruplex DNA substrates or disrupting protein-DNA complexes. The FANCJ-A349P allele failed to rescue cisplatin or telomestatin sensitivity of a FA-J null cell line as detected by cell survival or γ-H2AX foci formation. Furthermore, expression of FANCJ-A349P in a wild-type background exerted a dominant-negative effect, indicating that the mutant protein interferes with normal DNA metabolism. The ability of FANCJ to use the energy from ATP hydrolysis to produce the force required to unwind DNA or destabilize protein bound to DNA is required for its role in DNA repair.

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.

Kinetics of ATP hydrolysis during the DNA helicase II-promoted unwinding of duplex DNA

Biochemistry ◽  
1993 ◽  
Vol 32 (30) ◽  
pp. 7765-7771 ◽  
Author(s):  
Jaya G. Yodh ◽  
Floyd R. Bryant
Keyword(s):  
Atp Hydrolysis ◽  
Dna Helicase ◽  
Duplex Dna ◽  
Dna Helicase Ii ◽  
Kinetics Of
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