DNA excision by the SfiI restriction endonuclease

Nobbs, Timothy J.; Szczelkun, Mark D.; Wentzell, Lois M.; Halford, Stephen E.

doi:10.1006/jmbi.1998.1966

Cited by 59 publications

(77 citation statements)

References 40 publications

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“…We used our AFM data obtained for streptavidin tetramer (60 kDa) bound to biotinylated sites on DNA (13) to convert SfiI volume into molecular mass. Using the streptavidin tetramer volume (122 nm 3 ) the calculated molecular mass of SfiI in complex with DNA is 117 kDa very close to expected tetramer size of the protein (124 kDa) (6,17). This finding suggests that tetramer organization of the protein remains unchanged upon the preparation of the sample for AFM.…”

Section: Resultssupporting

confidence: 53%

DNA Strand Arrangement within the SfiI−DNA Complex: Atomic Force Microscopy Analysis

et al. 2005

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The SfiI restriction enzyme binds to DNA as a tetramer holding two usually distant DNA recognition sites together before a complete cleavage of four DNA strands. To elucidate structural properties of the SfiI-DNA complex, atomic force microscopy (AFM) imaging of the complexes under noncleaving reaction conditions (Ca 2+ instead of Mg 2+ in the reaction buffer) was performed. Intramolecular complexes formed by the interaction with two binding sites in one DNA molecule (cis interaction) as well as the complexes obtained by the interaction of two sites in different molecules (trans interaction) were analyzed. Complexes were identified unambiguously by the presence of a tall spherical blob at the DNA intersections. To characterize the path of DNA within the complex the angles between the DNA strands at the complex proximity regardless of the complex type were systematically analyzed. All the data show a clear-cut bimodal distributions centered around peak values corresponding to 60° and 120°. To unambiguously distinguish between the crossed and bent models for the DNA orientation within the complex, DNA templates with strands of different lengths and with different locations of the SfiI binding site were designed. The analysis of the AFM images for complexes of this type led to the conclusion that the DNA recognition sites within the complex are crossed. The angles 60° or 120° between the strands corresponds to complex in which one of the strands flipped the orientation relative to another. Both types of complexes for 5 different sequences in the center are present almost equally. This finding suggests that there is no preferential orientation of the DNA cognate site within the complex suggesting that the central part of the DNA binding site does not form strong sequence specific contacts with the protein. KeywordsDNA synaptic complexes; protein-DNA interaction; restriction-modification; DNA looping; scanning probe microscopy Restriction enzyme SfiI belongs to a family of type II restriction endonucleases that bind and cut two cognate sites (type IIf, e.g., article (1) and references therein). A few enzymes of type IIf enzymes have been analyzed (Bse634I, Cfr10I, NgoMIV, NaeI and SfiI) and some details shedding a light on the mechanisms of the actions have been revealed. SfiI has been studied extensively by Halford and coworkers (recent paper (2) and references therein). † This research was supported by Grant GM 062235 (YLL) from the National Institute of Health. Crystallographic data have been obtained for Bse634I (3), NgoMIV (4) and Cfr10I (5). SfiI binds as a homotetramer to two recognition sites on DNA duplexes at the recognition sequence 5'-GGCCNNNN^NGGCC-3' (N denotes any base and ^ marks a cleavage position) prior to cleavage (6). The recognition regions can be in one DNA molecule or in two different molecules (cis and trans position, respectively) with higher reaction rate for cis position due to the higher effective local concentration of the interacting sites for the intramolecular reaction versus ...

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Section: Resultssupporting

confidence: 53%

DNA Strand Arrangement within the SfiI−DNA Complex: Atomic Force Microscopy Analysis

et al. 2005

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“…Such behavior of SgrAI resembles the mode of action of tetrameric endonuclease SfiI that binds two copies of its recognition sequence before cleaving concertedly at four phosphodiester bonds (9,10). Other examples of restriction endonucleases that cleave two recognition sites in a concerted fashion are Cfr10I and NgoMIV (11,12).…”

Section: Discussionmentioning

confidence: 95%

“…When cleaving at the canonical sites, SgrAI also prefers the sites in cis, but operates on sites in trans at a 5-fold reduced rate (14). It has been shown that SfiI operates on sites either in cis or in trans, but cleaves sites in cis more efficiently simply because the sites in cis are closer to each other in the three-dimensional space (10). To cleave secondary sites in trans to canonical termini, SgrAI has to form a synapse of three elements-i.e., two termini and a secondary site-across three-dimensional space.…”

Section: Discussionmentioning

confidence: 99%

“…Recently, a similar reaction pathway has been shown to be characteristic for Sau3AI, except that it is a monomeric protein that dimerizes in the presence of DNA (8). SfiI (9,10), NgoMIV (11), and Cfr10I (12) are tetramers of identical subunits that need to bind to two recognition sites for catalysis, but in contrast to EcoRII or NaeI, they cleave four phosphodiester bonds in a concerted fashion (10)(11)(12). SgrAI, described here, is yet another example of the diverse mechanisms exhibited by restriction endonucleases.…”

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confidence: 95%

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Self-generated DNA termini relax the specificity of Sgr AI restriction endonuclease

Bitinaite

Schildkraut²

2002

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

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The primary target of SgrAI restriction endonuclease is a multiple sequence of the form 5 -CPu2CCGGPyG. Previous work had indicated that SgrAI must bind two recognition sites simultaneously for catalysis [Bilcock, D. T., Daniels, L. E., Bath, A. J. & Halford, S. E. (1999) J. Biol. Chem. 274, 36379 -36386]. In the present study, SgrAI is shown to cleave not only its canonical sequences, but also the sequences 5 -CPuCCGGPy(A,T,C) and 5 -CPuCCGGGG, both referred to as secondary sequences. On plasmid pSK7, SgrAI cleaves secondary sites 26-fold slower than the canonical site. However, the same plasmid, but without the canonical site, is cleaved 200-fold slower. We show that DNA termini generated by cleaving the canonical site for SgrAI assist in the cleavage of secondary sites. The SgrAI-termini in cis with respect to secondary site are markedly preferred over those in trans. The SgrAI-termini provided in a form of oligonucleotide duplex are also shown to stimulate canonical site cleavage. At a 40-fold molar excess of the SgrAI-termini over substrate, the SgrAI specificity is shown to improve by two orders of magnitude, because of concurrent 10-fold increase in the cleavage of canonical site and 50-fold decrease in the cleavage of secondary sites. The unconventional reaction pathway by which SgrAI utilizes the self-generated DNA termini to cleave its DNA targets has not been observed hitherto among type II restriction endonucleases. Based on our work and previous reports, a pathway of DNA binding and cleavage by the SgrAI restriction endonuclease is proposed.

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“…A number of conditions may be considered relevant in accordance with this, as follows. (i) REases of type II R-M systems usually function as homodimers, although one example of a homotetrameric REase has been described for SfiI (21). The dimerization or possibly multimerization step may permit a delay in restriction activity compared to methylation.…”

Section: Discussionmentioning

confidence: 99%

The Methyltransferase from the LlaDII Restriction-Modification System Influences the Level of Expression of Its Own Gene

Christensen

Josephsen

2004

J Bacteriol

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The type II restriction-modification (R-M) system LlaDII isolated from Lactococcus lactis contains two tandemly arranged genes, llaDIIR and llaDIIM, encoding a restriction endonuclease (REase) and a methyltransferase (MTase), respectively. Interestingly, two LlaDII recognition sites are present in the llaDIIM promoter region, suggesting that they may influence the activity of the promoter through methylation status. In this study, separate promoters for llaDIIR and llaDIIM were identified, and the regulation of the two genes at the transcriptional level was investigated. DNA fragments containing the putative promoters were cloned in a promoter probe vector and tested for activity in the presence and absence of the active MTase. The level of expression of the MTase was 5-to 10-fold higher than the level of expression of the REase. The results also showed that the presence of M.LlaDII reduced the in vivo expression of the llaDIIM promoter (P llaDIIM ) up to 1,000-fold, whereas the activity of the llaDIIR promoter (P llaDIIR ) was not affected. Based on site-specific mutations it was shown that both of the LlaDII recognition sites within P llaDIIM are required to obtain complete repression of transcriptional activity. No regulation was found for llaDIIR, which appears to be constitutively expressed.

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DNA excision by the SfiI restriction endonuclease

Cited by 59 publications

References 40 publications

DNA Strand Arrangement within the SfiI−DNA Complex: Atomic Force Microscopy Analysis

DNA Strand Arrangement within the SfiI−DNA Complex: Atomic Force Microscopy Analysis

Self-generated DNA termini relax the specificity of Sgr AI restriction endonuclease

The Methyltransferase from the LlaDII Restriction-Modification System Influences the Level of Expression of Its Own Gene

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