scholarly journals Staphylococcus aureus DinG, a helicase that has evolved into a nuclease

2012 ◽  
Vol 442 (1) ◽  
pp. 77-84 ◽  
Author(s):  
Anne-Marie McRobbie ◽  
Bjoern Meyer ◽  
Christophe Rouillon ◽  
Biljana Petrovic-Stojanovska ◽  
Huanting Liu ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Atp Hydrolysis ◽  
Nuclease Activity ◽  
Dna Helicase ◽  
Complementation Group ◽  
Helicase Domain ◽  
Dna And Rna ◽  
Ding Protein ◽  
Dna Strands ◽  
Binding Cleft

DinG (damage inducible gene G) is a bacterial superfamily 2 helicase with 5′→3′ polarity. DinG is related to the XPD (xeroderma pigmentosum complementation group D) helicase family, and they have in common an FeS (iron–sulfur)-binding domain that is essential for the helicase activity. In the bacilli and clostridia, the DinG helicase has become fused with an N-terminal domain that is predicted to be an exonuclease. In the present paper we show that the DinG protein from Staphylococcus aureus lacks an FeS domain and is not a DNA helicase, although it retains DNA-dependent ATP hydrolysis activity. Instead, the enzyme is an active 3′→5′ exonuclease acting on single-stranded DNA and RNA substrates. The nuclease activity can be modulated by mutation of the ATP-binding cleft of the helicase domain, and is inhibited by ATP or ADP, suggesting a modified role for the inactive helicase domain in the control of the nuclease activity. By degrading rather than displacing RNA or DNA strands, the S. aureus DinG nuclease may accomplish the same function as the canonical DinG helicase.

Association of human DNA helicase RecQ5β with RNA polymerase II and its possible role in transcription

2008 ◽  
Vol 413 (3) ◽  
pp. 505-516 ◽  
Author(s):  
Keiichi Izumikawa ◽  
Mitsuaki Yanagida ◽  
Toshiya Hayano ◽  
Hiroyuki Tachikawa ◽  
Wataru Komatsu ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Large Subunit ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Dna Helicase ◽  
Helicase Domain ◽  
Dna And Rna ◽  
Human Dna ◽  
Rnap Ii

Although RecQ5β is a ssDNA (single-stranded DNA)-stimulated ATPase and an ATP-dependent DNA helicase with strand-annealing activities, its cellular function remains to be explored. In the present paper, we used immunopurification and MS-based analyses to show that human DNA helicase RecQ5β is associated with at least four RNAP II (RNA polymerase II) subunits. RecQ5β was also present in complexes immunoprecipitated using three different antibodies against the large subunit of RNAP II, or in complexes immunoprecipitated using an anti-FLAG antibody against either FLAG–RNAP II 33 kDa subunit or FLAG–Pin1. Different regions of the non-helicase domain of the RecQ5β molecule were associated with hypophosphorylated and hyperphosphorylated forms of the RNAP II large subunit independently of DNA and RNA. RecQ5β was also found in nuclear chromatin fractions and associated with the coding regions of the LDL (low-density lipoprotein) receptor and β-actin genes. Knockdown of the RecQ5β transcript increased the transcription of those genes. The results of the present study suggest that RecQ5β has suppressive roles in events associated with RNAP II-dependent transcription.

scholarly journals Motif V regulates energy transduction between the flavivirus NS3 ATPase and RNA-binding cleft

2019 ◽  
Vol 295 (6) ◽  
pp. 1551-1564 ◽  
Author(s):  
Kelly E. Du Pont ◽  
Russell B. Davidson ◽  
Martin McCullagh ◽  
Brian J. Geiss
Keyword(s):  
Molecular Dynamics ◽  
Structural Changes ◽  
Rna Binding ◽  
Atp Hydrolysis ◽  
Nonstructural Protein ◽  
Binding Pocket ◽  
Energy Transduction ◽  
Helicase Domain ◽  
Genome Replication ◽  
Turnover Rates

The unwinding of dsRNA intermediates is critical for the replication of flavivirus RNA genomes. This activity is provided by the C-terminal helicase domain of viral nonstructural protein 3 (NS3). As a member of the superfamily 2 (SF2) helicases, NS3 requires the binding and hydrolysis of ATP/NTP to translocate along and unwind double-stranded nucleic acids. However, the mechanism of energy transduction between the ATP- and RNA-binding pockets is not well-understood. Previous molecular dynamics simulations conducted by our group have identified Motif V as a potential “communication hub” for this energy transduction pathway. To investigate the role of Motif V in this process, here we combined molecular dynamics, biochemistry, and virology approaches. We tested Motif V mutations in both the replicon and recombinant protein systems to investigate viral genome replication, RNA-binding affinity, ATP hydrolysis activity, and helicase-mediated unwinding activity. We found that the T407A and S411A substitutions in NS3 reduce viral replication and increase the helicase-unwinding turnover rates by 1.7- and 3.5-fold, respectively, suggesting that flaviviruses may use suboptimal NS3 helicase activity for optimal genome replication. Additionally, we used simulations of each mutant to probe structural changes within NS3 caused by each mutation. These simulations indicate that Motif V controls communication between the ATP-binding pocket and the helical gate. These results help define the linkage between ATP hydrolysis and helicase activities within NS3 and provide insight into the biophysical mechanisms for ATPase-driven NS3 helicase function.

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 Mutations in the WRN Gene in Mice Accelerate Mortality in a p53-Null Background

2000 ◽  
Vol 20 (9) ◽  
pp. 3286-3291 ◽  
Author(s):  
David B. Lombard ◽  
Caroline Beard ◽  
Brad Johnson ◽  
Robert A. Marciniak ◽  
Jessie Dausman ◽  
...  
Keyword(s):  
Mortality Rate ◽  
Life Span ◽  
Human Disease ◽  
Dna Helicase ◽  
Premature Aging ◽  
Mutant Mice ◽  
Helicase Domain ◽  
C Terminus ◽  
Accelerated Senescence

ABSTRACT Werner's syndrome (WS) is a human disease with manifestations resembling premature aging. The gene defective in WS, WRN, encodes a DNA helicase. Here, we describe the generation of mice bearing a mutation that eliminates expression of the C terminus of the helicase domain of the WRN protein. Mutant mice are born at the expected Mendelian frequency and do not show any overt histological signs of accelerated senescence. These mice are capable of living beyond 2 years of age. Cells from these animals do not show elevated susceptibility to the genotoxins camptothecin or 4-NQO. However, mutant fibroblasts senesce approximately one passage earlier than controls. Importantly,WRN−/− ;p53−/− mice show an increased mortality rate relative toWRN+/− ;p53−/− animals. We consider possible models for the synergy betweenp53 and WRN mutations for the determination of life span.

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 A Conserved Regulatory Module at the C Terminus of the Papillomavirus E1 Helicase Domain Controls E1 Helicase Assembly

2014 ◽  
Vol 89 (2) ◽  
pp. 1129-1142 ◽  
Author(s):  
Stephen Schuck ◽  
Arne Stenlund
Keyword(s):  
Inhibitory Effect ◽  
Dna Helicase ◽  
Regulatory Sequence ◽  
Helicase Domain ◽  
E1 Protein ◽  
C Terminus ◽  
Regulatory Module ◽  
Origin Of Dna Replication ◽  
Coding Capacity ◽  
Regulatory Challenges

ABSTRACTViruses frequently combine multiple activities into one polypeptide to conserve coding capacity. This strategy creates regulatory challenges to ascertain that the combined activities are compatible and do not interfere with each other. The papillomavirus E1 protein, as many other helicases, has the intrinsic ability to form hexamers and double hexamers (DH) that serve as the replicative DNA helicase. However, E1 also has the more unusual ability to generate local melting by forming a double trimer (DT) complex that can untwist the double-stranded origin of DNA replication (ori) DNA in preparation for DH formation. Here we describe a switching mechanism that allows the papillomavirus E1 protein to form these two different kinds of oligomers and to transition between them. We show that a conserved regulatory module attached to the E1 helicase domain blocks hexamer and DH formation and promotes DT formation. In the presence of the appropriate trigger, the inhibitory effect of the regulatory module is relieved and the transition to DH formation can occur.IMPORTANCEThis study provides a mechanistic understanding into how a multifunctional viral polypeptide can provide different, seemingly incompatible activities. A conserved regulatory sequence module attached to the AAA+helicase domain in the papillomavirus E1 protein allows the formation of different oligomers with different biochemical activities.

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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