PfoI, a unique type II restriction endonuclease that recognises the sequence 5'-TdownarrowCCNGGA-3'

Gaigalas, M.; Manelienė, Z.; Kazlauskienė, Rita; Petrušyté, M.; Janulaitis, A.

doi:10.1093/nar/gnf097

Cited by 4 publications

(5 citation statements)

References 6 publications

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“…Restriction endonuclease PfoI from P. fluorescens biovar 126 recognizes the interrupted hexanucleotide palindromic sequence 5′-TCCNGGA-3′ and cuts phosphodiester bonds on the 5′-sides of the outer cytosines ( 21 ). DNA cleavage by PfoI produces the same protruding pentanucleotide 5′-ends as Ecl18kI and the EcoRII-C/PspGI enzymes ( Table 1 ).…”

Section: Resultsmentioning

confidence: 99%

“…To express the PfoI protein cells were grown in LB medium with appropriate antibiotics at 30°C to an OD 600 of 0.7 and PfoI expression was induced by addition of arabinose to the final 0.2% concentration. The PfoI protein was purified as described previously ( 21 ). Fractions containing PfoI enzyme were pooled and dialysed against the storage buffer [10 mM Tris-HCl (pH 8.0 at 25°C), 300 mM KCl, 1 mM DTT, 1 mM EDTA (ethylenediaminetetraacetic acid), 50% glycerol] and stored at −20°C.…”

Section: Methodsmentioning

confidence: 99%

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Time-resolved fluorescence studies of nucleotide flipping by restriction enzymes

Neely

Тамулайтис

Chen

et al. 2009

Nucleic Acids Research

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Restriction enzymes Ecl18kI, PspGI and EcoRII-C, specific for interrupted 5-bp target sequences, flip the central base pair of these sequences into their protein pockets to facilitate sequence recognition and adjust the DNA cleavage pattern. We have used time-resolved fluorescence spectroscopy of 2-aminopurine-labelled DNA in complex with each of these enzymes in solution to explore the nucleotide flipping mechanism and to obtain a detailed picture of the molecular environment of the extrahelical bases. We also report the first study of the 7-bp cutter, PfoI, whose recognition sequence (T/CCNGGA) overlaps with that of the Ecl18kI-type enzymes, and for which the crystal structure is unknown. The time-resolved fluorescence experiments reveal that PfoI also uses base flipping as part of its DNA recognition mechanism and that the extrahelical bases are captured by PfoI in binding pockets whose structures are quite different to those of the structurally characterized enzymes Ecl18kI, PspGI and EcoRII-C. The fluorescence decay parameters of all the enzyme-DNA complexes are interpreted to provide insight into the mechanisms used by these four restriction enzymes to flip and recognize bases and the relationship between nucleotide flipping and DNA cleavage.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Time-resolved fluorescence studies of nucleotide flipping by restriction enzymes

Neely

Тамулайтис

Chen

et al. 2009

Nucleic Acids Research

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“…The first pipeline of this kind, RiceGAAS (Sakata et al, 2002) was developed originally for the annotation of the rice genome. Since then a few others have been established such as DNA subway (iPlant, USA) 11 , FPGP (Amano et al, 2010) and MAKER (Cantarel et al, 2008).…”

Section: Introductionmentioning

confidence: 99%

TriAnnot: A Versatile and High Performance Pipeline for the Automated Annotation of Plant Genomes

Leroy¹,

Guilhot²,

Sakai³

et al. 2012

Front. Plant Sci.

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In support of the international effort to obtain a reference sequence of the bread wheat genome and to provide plant communities dealing with large and complex genomes with a versatile, easy-to-use online automated tool for annotation, we have developed the TriAnnot pipeline. Its modular architecture allows for the annotation and masking of transposable elements, the structural, and functional annotation of protein-coding genes with an evidence-based quality indexing, and the identification of conserved non-coding sequences and molecular markers. The TriAnnot pipeline is parallelized on a 712 CPU computing cluster that can run a 1-Gb sequence annotation in less than 5 days. It is accessible through a web interface for small scale analyses or through a server for large scale annotations. The performance of TriAnnot was evaluated in terms of sensitivity, specificity, and general fitness using curated reference sequence sets from rice and wheat. In less than 8 h, TriAnnot was able to predict more than 83% of the 3,748 CDS from rice chromosome 1 with a fitness of 67.4%. On a set of 12 reference Mb-sized contigs from wheat chromosome 3B, TriAnnot predicted and annotated 93.3% of the genes among which 54% were perfectly identified in accordance with the reference annotation. It also allowed the curation of 12 genes based on new biological evidences, increasing the percentage of perfect gene prediction to 63%. TriAnnot systematically showed a higher fitness than other annotation pipelines that are not improved for wheat. As it is easily adaptable to the annotation of other plant genomes, TriAnnot should become a useful resource for the annotation of large and complex genomes in the future.

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“…Wild-type (WT) PfoI as well as mutant proteins were purified according to the protocol described in (19), with modifications. We carried out sequential chromatographies on HiTrap Heparin and MonoQ 5/50 columns (GE Healthcare) in the buffer containing 10 mM potassium phosphate pH 7.4, 1 mM Ethylenediaminetetraacetic acid ( EDTA), 100 mM NaCl and 5 mM β-mercaptoethanol.…”

Section: Methodsmentioning

confidence: 99%

“…PfoI, identified in Pseudomonas fluorescens biovar 126, recognizes the 7 bp sequence 5′-T|CCNGGA (19). PfoI shows no significant protein sequence similarity with CCGG-REs, except of few residues from the catalytic and CCGG recognition motifs that could be manually aligned (Figure 1A).…”

Section: Introductionmentioning

confidence: 99%

Unique mechanism of target recognition by PfoI restriction endonuclease of the CCGG-family

Tamulaitiene

Manakova

Jovaisaite

et al. 2018

Nucleic Acids Research

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Restriction endonucleases (REs) of the CCGG-family recognize a set of 4–8 bp target sequences that share a common CCGG or CCNGG core and possess PD…D/ExK nuclease fold. REs that interact with 5 bp sequence 5′-CCNGG flip the central N nucleotides and ‘compress’ the bound DNA to stack the inner base pairs to mimic the CCGG sequence. PfoI belongs to the CCGG-family and cleaves the 7 bp sequence 5′-T|CCNGGA ("|" designates cleavage position). We present here crystal structures of PfoI in free and DNA-bound forms that show unique active site arrangement and mechanism of sequence recognition. Structures and mutagenesis indicate that PfoI features a permuted E…ExD…K active site that differs from the consensus motif characteristic to other family members. Although PfoI also flips the central N nucleotides of the target sequence it does not ‘compress’ the bound DNA. Instead, PfoI induces a drastic change in DNA backbone conformation that shortens the distance between scissile phosphates to match that in the unperturbed CCGG sequence. Our data demonstrate the diversity and versatility of structural mechanisms employed by restriction enzymes for recognition of related DNA sequences.

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PfoI, a unique type II restriction endonuclease that recognises the sequence 5'-TdownarrowCCNGGA-3'

Abstract: A new type II restriction endonuclease designated PfoI has been partially purified from Pseudomonas fluorescens biovar 126. PfoI recognises the interrupted hexanucleotide palindromic sequence 5'-T downward arrow CCNGGA-3' and cleaves DNA to produce protruding pentanucleotide 5'-ends.

Cited by 4 publications

References 6 publications

Time-resolved fluorescence studies of nucleotide flipping by restriction enzymes

Time-resolved fluorescence studies of nucleotide flipping by restriction enzymes

TriAnnot: A Versatile and High Performance Pipeline for the Automated Annotation of Plant Genomes

Unique mechanism of target recognition by PfoI restriction endonuclease of the CCGG-family

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