scholarly journals Natural transformation protein ComFA exhibits single-stranded DNA translocase activity

2021 ◽  
Author(s):  
Hannah Reed Foster ◽  
Xiaoxuan Lin ◽  
Sriram Srikant ◽  
Rachel R Cueny ◽  
Tanya G Falbel ◽  
...  
Keyword(s):  
Atp Hydrolysis ◽  
Natural Transformation ◽  
Dna Unwinding ◽  
Dna Uptake ◽  
Single Stranded Dna ◽  
Bacterial Populations ◽  
Gram Positive Bacteria ◽  
Dead Box ◽  
Outstanding Question ◽  
Dna Translocase

Natural transformation is one of the major mechanisms of horizontal gene transfer in bacterial populations and has been demonstrated in numerous species of bacteria. Despite the prevalence of natural transformation, much of the molecular mechanism remains unexplored. One major outstanding question is how the cell powers DNA import, which is rapid and highly processive. ComFA is one of a handful of proteins required for natural transformation in gram-positive bacteria. Its structural resemblance to the DEAD-box helicase family has led to a long-held hypothesis that ComFA acts as a motor to help drive DNA import into the cytosol. Here, we explored the helicase and translocase activity of ComFA to address this hypothesis. We followed the DNA-dependent ATPase activity of ComFA and, combined with mathematical modeling, demonstrated that ComFA likely translocates on single-stranded DNA from 5′ to 3′. However, this translocase activity does not lead to DNA unwinding in the conditions we tested. Further, we analyzed the ATPase cycle of ComFA and found that ATP hydrolysis stimulates the release of DNA, providing a potential mechanism for translocation. These findings help define the molecular contribution of ComFA to natural transformation and support the conclusion that ComFA plays a key role in powering DNA uptake.

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 A Motif Unique to the Human Dead-Box Protein DDX3 Is Important for Nucleic Acid Binding, ATP Hydrolysis, RNA/DNA Unwinding and HIV-1 Replication

PLoS ONE ◽  
2011 ◽  
Vol 6 (5) ◽  
pp. e19810 ◽  
Author(s):  
Anna Garbelli ◽  
Sandra Beermann ◽  
Giulia Di Cicco ◽  
Ursula Dietrich ◽  
Giovanni Maga
Keyword(s):  
Nucleic Acid ◽  
Atp Hydrolysis ◽  
Dna Unwinding ◽  
Nucleic Acid Binding ◽  
Dead Box ◽  
Hiv 1

scholarly journals Human HELB is a processive motor protein which catalyses RPA clearance from single-stranded DNA

2021 ◽  
Author(s):  
Silvia Hormeno ◽  
Oliver J Wilkinson ◽  
Clara Aicart-Ramos ◽  
Sahiti Kuppa ◽  
Edwin Antony ◽  
...  
Keyword(s):  
Dna Replication ◽  
Direct Observation ◽  
Single Molecule ◽  
Atp Hydrolysis ◽  
Motor Protein ◽  
Dna Unwinding ◽  
Helicase Activity ◽  
Dna Loops ◽  
Single Stranded Dna ◽  
A Site

Human HELB is a poorly-characterised helicase suggested to play both positive and negative regulatory roles in DNA replication and recombination. In this work, we used bulk and single molecule approaches to characterise the biochemical activities of HELB protein with a particular focus on its interactions with RPA and RPA-ssDNA filaments. HELB is a monomeric protein which binds tightly to ssDNA with a site size of ~20 nucleotides. It couples ATP hydrolysis to translocation along ssDNA in the 5′-to-3′ direction accompanied by the formation of DNA loops and with an efficiency of 1 ATP per base. HELB also displays classical helicase activity but this is very weak in the absence of an assisting force. HELB binds specifically to human RPA which enhances its ATPase and ssDNA translocase activities but inhibits DNA unwinding. Direct observation of HELB on RPA nucleoprotein filaments shows that translocating HELB concomitantly clears RPA from single-stranded DNA.

scholarly journals Double-stranded DNA translocase activity of transcription factor TFIIH and the mechanism of RNA polymerase II open complex formation

2015 ◽  
Vol 112 (13) ◽  
pp. 3961-3966 ◽  
Author(s):  
James Fishburn ◽  
Eric Tomko ◽  
Eric Galburt ◽  
Steven Hahn
Keyword(s):  
Transcription Factor ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Atp Hydrolysis ◽  
Dna Unwinding ◽  
Pol Ii ◽  
Torsional Strain ◽  
Double Stranded Dna ◽  
Dna Translocase

Formation of the RNA polymerase II (Pol II) open complex (OC) requires DNA unwinding mediated by the transcription factor TFIIH helicase-related subunit XPB/Ssl2. Because XPB/Ssl2 binds DNA downstream from the location of DNA unwinding, it cannot function using a conventional helicase mechanism. Here we show that yeast TFIIH contains an Ssl2-dependent double-stranded DNA translocase activity. Ssl2 tracks along one DNA strand in the 5′ → 3′ direction, implying it uses the nontemplate promoter strand to reel downstream DNA into the Pol II cleft, creating torsional strain and leading to DNA unwinding. Analysis of the Ssl2 and DNA-dependent ATPase activity of TFIIH suggests that Ssl2 has a processivity of approximately one DNA turn, consistent with the length of DNA unwound during transcription initiation. Our results can explain why maintaining the OC requires continuous ATP hydrolysis and the function of TFIIH in promoter escape. Our results also suggest that XPB/Ssl2 uses this translocase mechanism during DNA repair rather than physically wedging open damaged DNA.

scholarly journals The Evolution of Bacterial Transformation: Sex With Poor Relations

Genetics ◽  
1997 ◽  
Vol 146 (1) ◽  
pp. 27-38 ◽  
Author(s):  
Rosemary J Redfield ◽  
Matthew R Schrag ◽  
Antony M Dean
Keyword(s):  
Natural Transformation ◽  
Deleterious Mutation ◽  
Fitness Function ◽  
Dna Uptake ◽  
Deleterious Mutations ◽  
Limited Data ◽  
Bacterial Populations ◽  
Selection For ◽  
Transformation Systems ◽  
Selection Of

Bacteria are the only organisms known to actively take up DNA and recombine it into their genomes. While such natural transformation systems may provide many of the same benefits that sexual reproduction provides eukaryotes, there are important differences that critically alter the consequences, especially when recombination's main benefit is reducing the mutation load. Here, analytical and numerical methods are used to study the selection of transformation genes in populations undergoing deleterious mutation. Selection for transformability depends on the shape of the fitness function against mutation. If the fitness function is linear, then transformation would be selectively neutral were it not for the possibility that transforming cells may take up DNA that converts them into nontransformable cells. If the selection includes strong positive (synergistic) epistasis, then transformation can be advantageous in spite of this risk. The effect of low quality DNA (from selectively killed cells) on selection is then studied analytically and found to impose an additional cost. The limited data available for real bacterial populations suggest that the conditions necessary for the evolution of transformation are unlikely to be met, and thus that DNA uptake may have some function other than recombination of deleterious mutations.

scholarly journals Antimicrobial Activity and Mode of Action of Celastrol, a Nortriterpen Quinone Isolated from Natural Sources

Foods ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 591
Author(s):  
Nayely Padilla-Montaño ◽  
Leandro de León Guerra ◽  
Laila Moujir
Keyword(s):  
Antimicrobial Activity ◽  
Cytoplasmic Membrane ◽  
Bactericidal Effect ◽  
Inoculum Size ◽  
Bacterial Populations ◽  
Gram Positive Bacteria ◽  
Dna And Rna ◽  
Dna And Rna Synthesis ◽  
Natural Derivative ◽  
Interesting Alternative

Species of the Celastraceae family are traditionally consumed in different world regions for their stimulating properties. Celastrol, a triterpene methylene quinone isolated from plants of celastraceas, specifically activates satiety centers in the brain that play an important role in controlling body weight. In this work, the antimicrobial activity and mechanism of action of celastrol and a natural derivative, pristimerin, were investigated in Bacillus subtilis. Celastrol showed a higher antimicrobial activity compared with pristimerin, being active against Gram-positive bacteria with minimum inhibitory concentrations (MICs) that ranged between 0.16 and 2.5 µg/mL. Killing curves displayed a bactericidal effect that was dependent on the inoculum size. Monitoring of macromolecular synthesis in bacterial populations treated with these compounds revealed inhibition in the incorporation of all radiolabeled precursors, but not simultaneously. Celastrol at 3 µg/mL and pristimerin at 10 µg/mL affected DNA and RNA synthesis first, followed by protein synthesis, although the inhibitory action on the uptake of radiolabeled precursors was more dramatic with celastrol. This compound also caused cytoplasmic membrane disruption observed by potassium leakage and formation of mesosome-like structures. The inhibition of oxygen consumption of whole and disrupted cells after treatments with both quinones indicates damage in the cellular structure, suggesting the cytoplasmic membrane as a potential target. These findings indicate that celastrol could be considered as an interesting alternative to control outbreaks caused by spore-forming bacteria.

scholarly journals DisA Limits RecG Activities at Stalled or Reversed Replication Forks

Cells ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1357
Author(s):  
Rubén Torres ◽  
Carolina Gándara ◽  
Begoña Carrasco ◽  
Ignacio Baquedano ◽  
Silvia Ayora ◽  
...  
Keyword(s):  
Atp Hydrolysis ◽  
Holliday Junctions ◽  
Genome Integrity ◽  
Stable Complex ◽  
Dna Unwinding ◽  
Replication Forks ◽  
Dna Structures ◽  
Checkpoint Protein

The DNA damage checkpoint protein DisA and the branch migration translocase RecG are implicated in the preservation of genome integrity in reviving haploid Bacillus subtilis spores. DisA synthesizes the essential cyclic 3′, 5′-diadenosine monophosphate (c‑di-AMP) second messenger and such synthesis is suppressed upon replication perturbation. In vitro, c-di-AMP synthesis is suppressed when DisA binds DNA structures that mimic stalled or reversed forks (gapped forks or Holliday junctions [HJ]). RecG, which does not form a stable complex with DisA, unwinds branched intermediates, and in the presence of a limiting ATP concentration and HJ DNA, it blocks DisA-mediated c-di-AMP synthesis. DisA pre-bound to a stalled or reversed fork limits RecG-mediated ATP hydrolysis and DNA unwinding, but not if RecG is pre-bound to stalled or reversed forks. We propose that RecG-mediated fork remodeling is a genuine in vivo activity, and that DisA, as a molecular switch, limits RecG-mediated fork reversal and fork restoration. DisA and RecG might provide more time to process perturbed forks, avoiding genome breakage.

How Chi Sequence Modifies RecBCD Single-Stranded DNA Translocase Activity

ChemPhysChem ◽  
2017 ◽  
Vol 19 (2) ◽  
pp. 243-247 ◽  
Author(s):  
Chia-Chuan Cho ◽  
Cinya Chung ◽  
Hung-Wen Li
Keyword(s):  
Single Stranded Dna ◽  
Chi Sequence ◽  
Dna Translocase

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 A DNase Encoded by Integrated Element CJIE1 Inhibits Natural Transformation of Campylobacter jejuni

2009 ◽  
Vol 191 (7) ◽  
pp. 2296-2306 ◽  
Author(s):  
Esther J. Gaasbeek ◽  
Jaap A. Wagenaar ◽  
Magalie R. Guilhabert ◽  
Marc M. S. M. Wösten ◽  
Jos P. M. van Putten ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Molecular Mechanism ◽  
Campylobacter Jejuni ◽  
Natural Transformation ◽  
Dnase Activity ◽  
Comparative Genome Hybridization ◽  
Dna Uptake ◽  
Knockout Mutant ◽  
Gene Encoding ◽  
Plasmid Analysis

ABSTRACT The species Campylobacter jejuni is considered naturally competent for DNA uptake and displays strong genetic diversity. Nevertheless, nonnaturally transformable strains and several relatively stable clonal lineages exist. In the present study, the molecular mechanism responsible for the nonnatural transformability of a subset of C. jejuni strains was investigated. Comparative genome hybridization indicated that C. jejuni Mu-like prophage integrated element 1 (CJIE1) was more abundant in nonnaturally transformable C. jejuni strains than in naturally transformable strains. Analysis of CJIE1 indicated the presence of dns (CJE0256), which is annotated as a gene encoding an extracellular DNase. DNase assays using a defined dns mutant and a dns-negative strain expressing Dns from a plasmid indicated that Dns is an endogenous DNase. The DNA-hydrolyzing activity directly correlated with the natural transformability of the knockout mutant and the dns-negative strain expressing Dns from a plasmid. Analysis of a broader set of strains indicated that the majority of nonnaturally transformable strains expressed DNase activity, while all naturally competent strains lacked this activity. The inhibition of natural transformation in C. jejuni via endogenous DNase activity may contribute to the formation of stable lineages in the C. jejuni population.

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