The Type I Restriction Endonuclease EcoR124I, Couples ATP Hydrolysis to Bidirectional DNA Translocation

Bianco, Piero R.; Hurley, Elizabeth M.

doi:10.1016/j.jmb.2005.07.055

Cited by 21 publications

(72 citation statements)

References 59 publications

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“…In addition, the presence of the antibody reduces bulk-phase ATPase activity by as much as 70% by an unknown mechanism. 19 If the hydrolysis of ATP is coupled to translocation as observed for other dsDNA translocases, 46 then the reduced rates of ATP hydrolysis for the antibody-tagged protein indicates that one would expect a reduced rate of translocation relative to the unmodified protein. In fact, the observed concentration rates are reduced by 70% compared to what we observe in this study.…”

Section: Resultsmentioning

confidence: 99%

Rad54 Oligomers Translocate and Cross-bridge Double-stranded DNA to Stimulate Synapsis

Bianco

Bradfield

Castanza

et al. 2007

Journal of Molecular Biology

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Rad54 is a key component of the eukaryotic recombination machinery. Its presence in DNA strand exchange reactions in vitro results in a significant stimulation in the overall reaction rate. Using untagged Rad54, we show that this stimulation can be attributed to enhancement of the formation of a key reaction intermediate known as DNA networks. Using a novel, single DNA molecule, dualoptical tweezers approach we show how Rad54 stimulates DNA network formation. We discovered that Rad54 oligomers possess a unique ability to cross-bridge or bind dsDNA molecules positioned in close proximity. Further, Rad54 oligomers rapidly translocate dsDNA while simultaneously inducing topological loops in the DNA at the locus of the oligomer. The combination of the crossbridging and dsDNA translocation activities of Rad54 stimulates the formation of DNA networks, leading to rapid and efficient DNA strand exchange by Rad51.

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Section: Resultsmentioning

confidence: 99%

Rad54 Oligomers Translocate and Cross-bridge Double-stranded DNA to Stimulate Synapsis

Bianco

Bradfield

Castanza

et al. 2007

Journal of Molecular Biology

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“…25 Heparin is similar to DNA as both molecules are highly charged linear polymers that behave as polyelectrolytes. 39 However, in contrast to DNA, it does not stimulate the ATPase activity of RecG (data not shown).…”

Section: Heparin Trapping Reveals That the Translocation Processivitymentioning

confidence: 99%

“…25 The conversion of ATP to ADP and P i is linked to the oxidation of NADH to NAD + , and is monitored as a decrease in absorbance at 340nm. Assays were performed at 37°C for 60 min and monitored using a Varian, Cary-50 Bio UV-visible spectrophotometer.…”

Section: Atp Hydrolysis Assaymentioning

confidence: 99%

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Characterization of the ATPase Activity of the Escherichia coli RecG Protein Reveals that the Preferred Cofactor is Negatively Supercoiled DNA

Slocum

Buss

Kimura

et al. 2007

Journal of Molecular Biology

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133

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RecG is a member of the superfamily 2 helicase family. Its possible role in vivo is ATP hydrolysis driven regression of stalled replication forks. To gain mechanistic insight into how this is achieved, a coupled spectrophotometric assay was utilized to characterize the ATPase activity of RecG in vitro. The results demonstrate an overwhelming preference for negatively supercoiled DNA ((-)scDNA) as a cofactor for the hydrolysis of ATP. In the presence of (-)scDNA the catalytic efficiency of RecG and the processivity (as revealed through heparin trapping), were higher than on any other cofactor examined. The activity of RecG on (-)scDNA was not due to the presence of single-stranded regions functioning as loading sites for the enzyme as relaxed circular DNA treated with DNA gyrase, resulted in the highest levels of ATPase activity. Relaxation of (-)scDNA by a topoisomerase resulted in a 12-fold decrease in ATPase activity, comparable to that observed on both linear double-stranded (ds)DNA and (+)scDNA. In addition to the elevated activity in the presence of (-)scDNA, RecG also has high activity on model 4Y-substrates (i.e. chicken foot structures). This is due largely to the high apparent affinity of the enzyme for this DNA substrate, which is 46-fold higher than a 2Y-substrate (i.e. a three-way with two single-stranded (ss)DNA arms). Finally, the enzyme exhibited significant, but lower activity on ssDNA. This activity was enhanced by the Escherichia coli stranded DNA-binding protein (SSB) protein, which occurs through stabilizing of the binding of RecG to ssDNA. Stabilization is not afforded by the bacteriophage gene 32 protein, indicating a species specific, protein-protein interaction is involved. These results combine to provide significant insight into the manner and timing of the interaction of RecG with DNA at stalled replication forks.

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“…This system encodes a methylase that is also responsible for sequence specificity and an endonuclease responsible for cleavage of unmethylated DNA. The type I and type III restriction endonucleases harbor a superfamily 2 helicase domain (16)(17)(18), which is required for translocation on dsDNA (17,19). Finally, a fourth restriction system cleaves DNA when it contains modified cytosine residues (20).…”

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A type III-like restriction endonuclease functions as a major barrier to horizontal gene transfer in clinical Staphylococcus aureus strains

Corvaglia

François

Hernández

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

113

109

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Staphylococcus aureus is an versatile pathogen that can cause lifethreatening infections. Depending on the clinical setting, up to 50% of S. aureus infections are caused by methicillin-resistant strains (MRSA) that in most cases are resistant to many other antibiotics, making treatment difficult. The emergence of communityacquired MRSA drastically changed the picture by increasing the risk of MRSA infections. Horizontal transfer of genes encoding for antibiotic resistance or virulence factors is a major concern of multidrug-resistant S. aureus infections and epidemiology. We identified and characterized a type III-like restriction system present in clinical S. aureus strains that prevents transformation with DNA from other bacterial species. Interestingly, our analysis revealed that some clinical MRSA strains are deficient in this restriction system, and thus are hypersusceptible to the horizontal transfer of DNA from other species, such as Escherichia coli, and could easily acquire a vancomycin-resistance gene from enterococci. Inactivation of this restriction system dramatically increases the transformation efficiency of clinical S. aureus strains, opening the field of molecular genetic manipulation of these strains using DNA of exogenous origin.antibiotic resistance | transformation | targetron

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The Type I Restriction Endonuclease EcoR124I, Couples ATP Hydrolysis to Bidirectional DNA Translocation

Cited by 21 publications

References 59 publications

Rad54 Oligomers Translocate and Cross-bridge Double-stranded DNA to Stimulate Synapsis

Rad54 Oligomers Translocate and Cross-bridge Double-stranded DNA to Stimulate Synapsis

Characterization of the ATPase Activity of the Escherichia coli RecG Protein Reveals that the Preferred Cofactor is Negatively Supercoiled DNA

A type III-like restriction endonuclease functions as a major barrier to horizontal gene transfer in clinical Staphylococcus aureus strains

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