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A putative mobile genetic element carrying a novel type IIF restriction-modification system (PluTI).

Khan F, Furuta Y, Kawai M, Kaminska KH, Ishikawa K, Bujnicki JM, Kobayashi I - Nucleic Acids Res. (2010)

Bottom Line: Subsequently, the protein, named R.PluTI, was expressed in vivo, purified and found to be a novel type IIF restriction enzyme that recognizes 5'-GGCGC/C-3' (/ indicates position of cleavage).These results suggested that they constitute a restriction-modification system, present on the putative mobile element.Our approach thus allowed detection of a previously uncharacterized family of DNA-interacting proteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Genome Sciences, Graduate School of Frontier Sciences, University of Tokyo, Japan.

ABSTRACT
Genome comparison and genome context analysis were used to find a putative mobile element in the genome of Photorhabdus luminescens, an entomopathogenic bacterium. The element is composed of 16-bp direct repeats in the terminal regions, which are identical to a part of insertion sequences (ISs), a DNA methyltransferase gene homolog, two genes of unknown functions and an open reading frame (ORF) (plu0599) encoding a protein with no detectable sequence similarity to any known protein. The ORF (plu0599) product showed DNA endonuclease activity, when expressed in a cell-free expression system. Subsequently, the protein, named R.PluTI, was expressed in vivo, purified and found to be a novel type IIF restriction enzyme that recognizes 5'-GGCGC/C-3' (/ indicates position of cleavage). R.PluTI cleaves a two-site supercoiled substrate at both the sites faster than a one-site supercoiled substrate. The modification enzyme homolog encoded by plu0600, named M.PluTI, was expressed in Escherichia coli and shown to protect DNA from R.PluTI cleavage in vitro, and to suppress the lethal effects of R.PluTI expression in vivo. These results suggested that they constitute a restriction-modification system, present on the putative mobile element. Our approach thus allowed detection of a previously uncharacterized family of DNA-interacting proteins.

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R.PluTI reactions on one- and two-site plasmids. Reaction contained 0.5 nM of purified R.PluTI and 4 nM of supercoiled (SC) DNA in NEB1* buffer (10 mM Tris–propane buffer, pH 7.0 supplemented with 10 mM MgCl2, 1 mM dithiothreitol and 100 µg/ml BSA) at 37°C. DNA was (a and b), LITMUS 38i, with one R.PluTI site; (c and d), pFK1 with two R.PluTI sites. (a and c) Agarose gel electrogram of DNA taken from the reaction at the indicated time: electrophoretic mobilities of open-circle (OC), full-length linear (FLL), supercoiled (SC) and reference DNAs (1.3 kb linear fragment) are marked on the right, in (c) two linear products (L1 and L2) resulting from cutting at the both sites are also marked. (b and d) Concentrations of substrate (SC) and products (OC, FLL and average of L1, L2) over 120 min of reaction. Error bars (shown only one side, and in some cases masked by symbols) indicate standard deviation from three or more independent sets of experiments.
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Figure 3: R.PluTI reactions on one- and two-site plasmids. Reaction contained 0.5 nM of purified R.PluTI and 4 nM of supercoiled (SC) DNA in NEB1* buffer (10 mM Tris–propane buffer, pH 7.0 supplemented with 10 mM MgCl2, 1 mM dithiothreitol and 100 µg/ml BSA) at 37°C. DNA was (a and b), LITMUS 38i, with one R.PluTI site; (c and d), pFK1 with two R.PluTI sites. (a and c) Agarose gel electrogram of DNA taken from the reaction at the indicated time: electrophoretic mobilities of open-circle (OC), full-length linear (FLL), supercoiled (SC) and reference DNAs (1.3 kb linear fragment) are marked on the right, in (c) two linear products (L1 and L2) resulting from cutting at the both sites are also marked. (b and d) Concentrations of substrate (SC) and products (OC, FLL and average of L1, L2) over 120 min of reaction. Error bars (shown only one side, and in some cases masked by symbols) indicate standard deviation from three or more independent sets of experiments.

Mentions: This mechanism is distinguished by steady-state cleavage reactions when enzyme concentration is considerably lower than the substrate. Based on the one-site plasmid, LITMUS38i, we constructed a two-site plasmid, pFK1 with two R.PluTI sites placed 853 bp away from each other, with identical flanking sequences (Supplementary Figure S6). R.PluTI consumed initial 10% substrate at a rate of 0.011 nM/min for the one-site substrate, and 0.28 nM/min for the two-site substrate (Figure 3). In the reaction on a SC one-site plasmid, OC form that was cut only at one strand was generated, along with a relatively small fraction of FLL form cut on both strands (Figure 3a–b). In contrast, products from the two-site plasmid were mainly linear fragments L1 and L2, which were formed by cleavage of both strands at both sites (Figure 3c–d). Less amounts of the FLL and OC forms were obtained, indicating concerted cleavage mechanism working at both sites. These kinetics were very similar to those of NgoMIV, a Type IIF enzyme (19).Figure 3.


A putative mobile genetic element carrying a novel type IIF restriction-modification system (PluTI).

Khan F, Furuta Y, Kawai M, Kaminska KH, Ishikawa K, Bujnicki JM, Kobayashi I - Nucleic Acids Res. (2010)

R.PluTI reactions on one- and two-site plasmids. Reaction contained 0.5 nM of purified R.PluTI and 4 nM of supercoiled (SC) DNA in NEB1* buffer (10 mM Tris–propane buffer, pH 7.0 supplemented with 10 mM MgCl2, 1 mM dithiothreitol and 100 µg/ml BSA) at 37°C. DNA was (a and b), LITMUS 38i, with one R.PluTI site; (c and d), pFK1 with two R.PluTI sites. (a and c) Agarose gel electrogram of DNA taken from the reaction at the indicated time: electrophoretic mobilities of open-circle (OC), full-length linear (FLL), supercoiled (SC) and reference DNAs (1.3 kb linear fragment) are marked on the right, in (c) two linear products (L1 and L2) resulting from cutting at the both sites are also marked. (b and d) Concentrations of substrate (SC) and products (OC, FLL and average of L1, L2) over 120 min of reaction. Error bars (shown only one side, and in some cases masked by symbols) indicate standard deviation from three or more independent sets of experiments.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2875022&req=5

Figure 3: R.PluTI reactions on one- and two-site plasmids. Reaction contained 0.5 nM of purified R.PluTI and 4 nM of supercoiled (SC) DNA in NEB1* buffer (10 mM Tris–propane buffer, pH 7.0 supplemented with 10 mM MgCl2, 1 mM dithiothreitol and 100 µg/ml BSA) at 37°C. DNA was (a and b), LITMUS 38i, with one R.PluTI site; (c and d), pFK1 with two R.PluTI sites. (a and c) Agarose gel electrogram of DNA taken from the reaction at the indicated time: electrophoretic mobilities of open-circle (OC), full-length linear (FLL), supercoiled (SC) and reference DNAs (1.3 kb linear fragment) are marked on the right, in (c) two linear products (L1 and L2) resulting from cutting at the both sites are also marked. (b and d) Concentrations of substrate (SC) and products (OC, FLL and average of L1, L2) over 120 min of reaction. Error bars (shown only one side, and in some cases masked by symbols) indicate standard deviation from three or more independent sets of experiments.
Mentions: This mechanism is distinguished by steady-state cleavage reactions when enzyme concentration is considerably lower than the substrate. Based on the one-site plasmid, LITMUS38i, we constructed a two-site plasmid, pFK1 with two R.PluTI sites placed 853 bp away from each other, with identical flanking sequences (Supplementary Figure S6). R.PluTI consumed initial 10% substrate at a rate of 0.011 nM/min for the one-site substrate, and 0.28 nM/min for the two-site substrate (Figure 3). In the reaction on a SC one-site plasmid, OC form that was cut only at one strand was generated, along with a relatively small fraction of FLL form cut on both strands (Figure 3a–b). In contrast, products from the two-site plasmid were mainly linear fragments L1 and L2, which were formed by cleavage of both strands at both sites (Figure 3c–d). Less amounts of the FLL and OC forms were obtained, indicating concerted cleavage mechanism working at both sites. These kinetics were very similar to those of NgoMIV, a Type IIF enzyme (19).Figure 3.

Bottom Line: Subsequently, the protein, named R.PluTI, was expressed in vivo, purified and found to be a novel type IIF restriction enzyme that recognizes 5'-GGCGC/C-3' (/ indicates position of cleavage).These results suggested that they constitute a restriction-modification system, present on the putative mobile element.Our approach thus allowed detection of a previously uncharacterized family of DNA-interacting proteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Genome Sciences, Graduate School of Frontier Sciences, University of Tokyo, Japan.

ABSTRACT
Genome comparison and genome context analysis were used to find a putative mobile element in the genome of Photorhabdus luminescens, an entomopathogenic bacterium. The element is composed of 16-bp direct repeats in the terminal regions, which are identical to a part of insertion sequences (ISs), a DNA methyltransferase gene homolog, two genes of unknown functions and an open reading frame (ORF) (plu0599) encoding a protein with no detectable sequence similarity to any known protein. The ORF (plu0599) product showed DNA endonuclease activity, when expressed in a cell-free expression system. Subsequently, the protein, named R.PluTI, was expressed in vivo, purified and found to be a novel type IIF restriction enzyme that recognizes 5'-GGCGC/C-3' (/ indicates position of cleavage). R.PluTI cleaves a two-site supercoiled substrate at both the sites faster than a one-site supercoiled substrate. The modification enzyme homolog encoded by plu0600, named M.PluTI, was expressed in Escherichia coli and shown to protect DNA from R.PluTI cleavage in vitro, and to suppress the lethal effects of R.PluTI expression in vivo. These results suggested that they constitute a restriction-modification system, present on the putative mobile element. Our approach thus allowed detection of a previously uncharacterized family of DNA-interacting proteins.

Show MeSH
Related in: MedlinePlus