EcoRV catalysis with a pre-bent substrate

Sanchez, Daniel S.; Zocchi, Giovanni

doi:10.1063/1.4921837

“…In this method, two single strands of DNA of different lengths (45 bases and 30 bases) form a circular bent-DNA molecule upon hybridization (Fig. 4a) (61)(62)(63)(64)(65)(66)(67). Stresses in the circular DNA due to the bending of the double-stranded segment make the molecule more prone to perturbations and, thus, to amplified interactions between the DNA and other molecules; therefore, these molecules are referred to as "amplifiers" (60).…”

Section: Resultsmentioning

confidence: 99%

Silver Ions Caused Faster Diffusive Dynamics of Histone-Like Nucleoid-Structuring Proteins in Live Bacteria

Sadoon

¹

,

Khadka

²

,

Freeland

³

et al. 2020

Appl Environ Microbiol

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The antimicrobial activity and mechanism of silver ions (Ag+) have gained broad attention in recent years. However, dynamic studies are rare in this field. Here, we report our measurement of the effects of Ag+ ions on the dynamics of histone-like nucleoid-structuring (H-NS) proteins in live bacteria using single-particle-tracking photoactivated localization microscopy (sptPALM). It was found that treating the bacteria with Ag+ ions led to faster diffusive dynamics of H-NS proteins. Several techniques were used to understand the mechanism of the observed faster dynamics. Electrophoretic mobility shift assay on purified H-NS proteins indicated that Ag+ ions weaken the binding between H-NS proteins and DNA. Isothermal titration calorimetry confirmed that DNA and Ag+ ions interact directly. Our recently developed sensing method based on bent DNA suggested that Ag+ ions caused dehybridization of double-stranded DNA (i.e., dissociation into single strands). These evidences led us to a plausible mechanism for the observed faster dynamics of H-NS proteins in live bacteria when subjected to Ag+ ions: Ag+-induced DNA dehybridization weakens the binding between H-NS proteins and DNA. This work highlighted the importance of dynamic study of single proteins in live cells for understanding the functions of antimicrobial agents in bacteria. IMPORTANCE As so-called “superbug” bacteria resistant to commonly prescribed antibiotics have become a global threat to public health in recent years, noble metals, such as silver, in various forms have been attracting broad attention due to their antimicrobial activities. However, most of the studies in the existing literature have relied on the traditional bioassays for studying the antimicrobial mechanism of silver; in addition, temporal resolution is largely missing for understanding the effects of silver on the molecular dynamics inside bacteria. Here, we report our study of the antimicrobial effect of silver ions at the nanoscale on the diffusive dynamics of histone-like nucleoid-structuring (H-NS) proteins in live bacteria using single-particle-tracking photoactivated localization microscopy. This work highlights the importance of dynamic study of single proteins in live cells for understanding the functions of antimicrobial agents in bacteria.

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“…To overcome this challenge to test our hypothesis, we exploited our recently developed method using bent DNA molecules [59] . In this method, two single strands of DNA of different lengths (45 bases and 30 bases) form a circular bent DNA molecule upon hybridization [60][61][62][63][64][65][66] (Fig. 4a).…”

Section: Dehybridization Of Bent Duplex Dna Induced By Ag + Ionsmentioning

confidence: 99%

Faster diffusive dynamics of histone-like nucleoid structuring proteins in live bacteria caused by silver ions

Sadoon

¹

,

Khadka

²

,

Freeland

³

et al. 2019

Preprint

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The antimicrobial activity and mechanism of silver ions (Ag + ) have gained broad attention in recent years. However, dynamic studies are rare in this field. Here, we report our measurement of the effects of Ag + ions on the dynamics of histone-like nucleoid structuring (H-NS) proteins in live bacteria using single-particle tracking photoactivated localization microscopy (sptPALM). It was found that treating the bacteria with Ag + ions led to faster diffusive dynamics of H-NS proteins. Several techniques were used to understand the mechanism of the observed faster dynamics. Electrophoretic mobility shift assay on purified H-NS proteins indicated that Ag + ions weaken the binding between H-NS proteins and DNA. Isothermal titration calorimetry confirmed that DNA and Ag + ions interact directly. Our recently developed sensing method based on bent DNA suggested that Ag + ions caused dehybridization of double-stranded DNA (i.e., dissociation into single strands). These evidences led us to a plausible mechanism for the observed faster dynamics of H-NS proteins in live bacteria when subjected to Ag + ions: Ag + -induced DNA dehybridization weakens the binding between H-NS proteins and DNA. This work highlighted the importance of dynamic study of single proteins in the live cells for understanding the functions of antimicrobial agents to the bacteria.

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“…A recent experimental study on EcoRV‐DNA complex shows that the speed of the enzymatic reaction can be modulated by mechanically stressing the DNA substrate. [ 30 ] The bent DNA conformations in the crystal structures in presence of both Mg 2+ and Ca 2+ , rule out that the overall DNA conformations account for the specificity of cofactor Mg 2+ vis‐à‐vis Ca 2+ . [ 18–20 ] This, however, does not rule out the possibility of sensitivity of fluctuations of microscopic conformational variables [ 31 ] to the type and locations of the metal ions.…”

Section: Introductionmentioning

confidence: 99%

Microscopic insight to specificity of metal ion cofactor in DNA cleavage by restriction endonuclease EcoRV

Mandal

¹

,

Maganti

²

,

Mondal

³

et al. 2020

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Restriction endonucleases protect bacterial cells against bacteriophage infection by cleaving the incoming foreign DNA into fragments. In presence of Mg 2+ ions, EcoRV is able to cleave the DNA but not in presence of Ca 2+ , although the protein binds to DNA in presence of both metal ions. We make an attempt to understand this difference using conformational thermodynamics. We calculate the changes in conformational free energy and entropy of conformational degrees of freedom, like DNA base pair steps and dihedral angles of protein residues in Mg 2+ (A)-EcoRV-DNA complex compared to Ca 2+ (S)-EcoRV-DNA complex using all-atom molecular dynamics (MD) trajectories of the complexes. We find that despite conformational stability and order in both complexes, the individual degrees of freedom behave differently in the presence of two different metal ions. The base pairs in cleavage region are highly disordered in Ca 2+ (S)-EcoRV-DNA compared to Mg 2+ (A)-EcoRV-DNA. One of the acidic residues ASP90, coordinating to the metal ion in the vicinity of the cleavage site, is conformationally destabilized and disordered, while basic residue LYS92 gets conformational stability and order in Ca 2+ (S) bound complex than in Mg 2+ (A) bound complex. The enhanced fluctuations hinder placement of the metal ion in the vicinity of the scissile phosphate of DNA. Similar loss of conformational stability and order in the cleavage region is observed by the replacement of the metal ion. Considering the placement of the metal ion near scissile phosphate as requirement for cleavage action, our results suggest that the changes in conformational stability and order of the base pair steps and the protein residues lead to cofactor sensitivity of the enzyme. Our method based on fluctuations of microscopic conformational variables can be applied to understand enzyme activities in other protein-DNA systems.

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EcoRV catalysis with a pre-bent substrate

Cited by 3 publications

References 30 publications

Silver Ions Caused Faster Diffusive Dynamics of Histone-Like Nucleoid-Structuring Proteins in Live Bacteria

Silver Ions Caused Faster Diffusive Dynamics of Histone-Like Nucleoid-Structuring Proteins in Live Bacteria

Faster diffusive dynamics of histone-like nucleoid structuring proteins in live bacteria caused by silver ions

Microscopic insight to specificity of metal ion cofactor in DNA cleavage by restriction endonuclease EcoRV

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