Identification and characterization of a new HNH restriction endonuclease with unusual properties

Santoshi, Meghna; Engleng, Bharat; Eligar, Sachin M.; Ratnakar, Immadi Siva; Nagamalleshwari, Easa; Nagaraja, Valakunja

doi:10.1007/s00253-023-12717-8

Cited by 2 publications

(3 citation statements)

References 45 publications

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“…It is also of note that Pa DpsL, like Pa Dps ( Rajapaksha et al, 2023 ), can efficiently nick DNA. In this context, restriction-modification systems, consisting of a restriction endonuclease and cognate methyl transferase, are considered innate immune mechanisms that protect their host against foreign DNA ( Santoshi et al, 2023 ). Nicking endonucleases, like restriction endonucleases, recognize a short specific sequence in double-stranded DNA and cleave it at a specific position relative to the recognition sequence.…”

Section: Discussionmentioning

confidence: 99%

Pseudomonas aeruginosa gene PA4880 encodes a Dps-like protein with a Dps fold, bacterioferritin-type ferroxidase centers, and endonuclease activity

Rajapaksha,

Yao,

Cook

et al. 2024

Front. Mol. Biosci.

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We report the biochemical, structural, and functional characterization of the protein coded by gene PA4880 in the P. aeruginosa PAO1 genome. The PA4880 gene had been annotated as coding a probable bacterioferritin. Our structural work shows that the product of gene PA4880 is a protein that adopts the Dps subunit fold, which oligomerizes into a 12-mer quaternary structure. Unlike Dps, however, the ferroxidase di-iron centers and iron coordinating ligands are buried within each subunit, in a manner identical to that observed in the ferroxidase center of P. aeruginosa bacterioferritin. Since these structural characteristics correspond to Dps-like proteins, we term the protein as P. aeruginosa Dps-like, or Pa DpsL. The ferroxidase centers in Pa DpsL catalyze the oxidation of Fe2+ utilizing O2 or H2O2 as oxidant, and the resultant Fe3+ is compartmentalized in the interior cavity. Interestingly, incubating Pa DpsL with plasmid DNA results in efficient nicking of the DNA and at higher concentrations of Pa DpsL the DNA is linearized and eventually degraded. The nickase and endonuclease activities suggest that Pa DpsL, in addition to participating in the defense of P. aeruginosa cells against iron-induced toxicity, may also participate in the innate immune mechanisms consisting of restriction endonucleases and cognate methyl transferases.

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Section: Discussionmentioning

confidence: 99%

Pseudomonas aeruginosa gene PA4880 encodes a Dps-like protein with a Dps fold, bacterioferritin-type ferroxidase centers, and endonuclease activity

Rajapaksha,

Yao,

Cook

et al. 2024

Front. Mol. Biosci.

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“…It was shown that despite PaqCI and FokI sharing similar structural mechanisms of DNA cleavage, these enzymes have considerable differences in their domain organization and quaternary architecture. Another member of this subtype, KpnK, can be considered a recently described enzyme, which was found in pathogenic Klebsiella pneumonia [90]. This enzyme is a monomer in solution, nicks double-stranded DNA, recognizes degenerate Structural data obtained for complexes of FokI with a 20 bp DNA fragment containing the recognition site (5 -GGATG-3 ) have revealed additional distinctive features of this enzyme.…”

Section: Endonucleases Of Subtype Iismentioning

confidence: 99%

“…In other cases, for example, for MnlI [100], it was shown that cleavage of single-stranded DNA proceeds in the presence of Ni 2+ and some other transition metal ions, whereas Mg 2 + ions are required for the cleavage of double-stranded DNA. Moreover, nicking endonucleases [101][102][103] become increasingly attractive for their application in single-strand cleavage of double-stranded DNA [104][105][106][107], and the list of these enzymes is regularly updated with new natural and artificial members [90,108,109]. Indeed, nicking endonucleases have been used in DNA optical mapping by nicking, nick translation of the nicked sites with fluorescently labeled dNTP, and religation of the nicked strand [110,111].…”

Section: Single-stranded Dna Cleavagementioning

confidence: 99%

Historical Aspects of Restriction Endonucleases as Intelligent Scissors for Genetic Engineering

Alekseeva,

Kuznetsov

2023

Fermentation

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Restriction endonucleases are a component of restriction–modification systems, where the main biological function is to protect bacterial cells from incoming foreign DNA molecules. There are four main types of restriction enzymes (types I, II, III, and IV), which differ in protein composition, cofactor requirements, and mode of action. The most studied are representatives of type II, which specifically recognize DNA sequences of 4–8 bp and catalyze DNA cleavage within these sequences or not far from them. The exceptional precision of type II enzymes has made them indispensable for DNA manipulations. Although hundreds of DNA restriction enzymes are currently known, there is still a need for enzymes that recognize new DNA targets. For this reason, the discovery of new natural restriction endonucleases and rational design of their properties (to obtain enzymes with high specificity for a unique nucleotide sequence at a restriction site and without nonspecific activity) will expand the list of enzymes for use in biotechnology and genetic engineering. This review briefly touches upon the main types of restriction endonucleases, their classification, nomenclature, and typical properties, and it concisely describes approaches to the construction of enzymes with altered properties.

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Identification and characterization of a new HNH restriction endonuclease with unusual properties

Cited by 2 publications

References 45 publications

Pseudomonas aeruginosa gene PA4880 encodes a Dps-like protein with a Dps fold, bacterioferritin-type ferroxidase centers, and endonuclease activity

Pseudomonas aeruginosa gene PA4880 encodes a Dps-like protein with a Dps fold, bacterioferritin-type ferroxidase centers, and endonuclease activity

Historical Aspects of Restriction Endonucleases as Intelligent Scissors for Genetic Engineering

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