Digestion conditions resulting in altered cut site specificity for<i>Hinf</i>l

Kriss, Jodi; Herrin, George; Forman, Lisa; Cotton, Robin W.

doi:10.1093/nar/18.12.3665

Cited by 8 publications

(5 citation statements)

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“…Under star conditions sites are also cleaved that deviate from the cognate sites by one base pair (EcoRI: Gardner et al, 1982). Alkaline pH, substitution of Mg" by Mn2+, and presence of organic solvents are also star conditions for several other restriction endonucleases (BamHI : Xu and Schildkraut, 1991 b;BsuR1: Heininger et al, 1977;CeqI: Izsvak and Duda, 1989;DdeI: Makula and Meagher, 1980;EcoRV: Halford et al, 1986;HindIII: Hsu and Berg, 1978;HinfI: Petronzino and Schildkraut, 1990;Kriss et al, 1990;MboII: Sektas et al, 1995;PstI: Malyguine and Vannier, 1980;PvuII: Nasri and Thomas, 1987;RsrI: Aiken and Gumport, 1988;SulI: Malyguine and Vannier, 1980;TaqI: Barany, 1988;Caoet al, 1995;TthlllI: Shinomiya et a]., 1982).…”

Section: Mechanism Of Dna Cleavagementioning

confidence: 99%

Recognition and Cleavage of DNA by Type‐II Restriction Endonucleases

Pingoud

Jeltsch

1997

European Journal of Biochemistry

321

259

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Restriction endonucleases are enzymes which recognize short DNA sequences and cleave the DNA in both strands. Depending on the enzymological properties different types are distinguished. Type I1 restriction endonucleases are homodimers which recognize short palindromic sequences 4-8 bp in length and, in the presence of Mg", cleave the DNA within or next to the recognition site. They are capable of non-specific binding to DNA and make use of linear diffusion to locate their target site. Binding and recognition of the specific site involves contacts to the bases of the recognition sequence and the phosphodiester backbone over approximately 10-12 bp. In general, recognition is highly redundant which explains the extreme specificity of these enzymes. Specific binding is accompanied by conformational changes over both the protein and the DNA. This mutual induced fit leads to the activation of the catalytic centers. The precise mechanism of cleavage has not yet been established for any restriction endonuclease. Currently two models are discussed: the substrate-assisted catalysis mechanism and the two-metal-ion mechanism. Structural similarities identified between EcoRI, EcoRV, BarnHI, PvuII and Cfrl 01 suggest that many type I1 restriciton endonucleases are not only functionally but also evolutionarily related.Keywords: protein -nucleic-acid interaction ; restriction-modification system ; restriction endonuclease ; DNA methyltransferase; non-specific DNA binding ; facilitated diffusion; specific DNA binding ; DNA recognition; phosphodiester bond cleavage ; catalytic mechanism. Overview of restriction endonucleasesRestriction endonucleases are parts of restriction-modification (RM) systems which occur ubiquitously among prokaryotic organisms. RM systems comprise an endonuclease and a methyltransferase activity. They serve to protect bacterial cells against bacteriophage infection, because incoining foreign DNA is highly specifically cleaved by the restriction enzyme if it contains the recognition sequence of the endonuclease. The cellular DNA is protected from cleavage by a specific methylation within the recognition sequence, which is introduced by the methyltransferase (reviews : Noyer-Weidner and Trautner, 1993 ;Cheng, 1995). RM systems can be divided into different types according to the subunit composition and the cofactor requirement (Table 1) (reviews: Wilson, 1991 ;Wilson and Murray, 1991;Heitman, 1993). Type I RM systems consist of three subunits: R for restriction, M for modification and S for specificity. They usually form pentameric complexes comprising two R, two M and one S subunit which recognizes a bipartite recognition sequence. Modification requires the presence of S-adenosyl-methionine (AdoMet) and occurs within the recognition sequence, while cleavage takes place at distant random sites and requires AdoMet as well as ATP which is hydrolyzed in large amounts

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Section: Mechanism Of Dna Cleavagementioning

confidence: 99%

Recognition and Cleavage of DNA by Type‐II Restriction Endonucleases

Pingoud

Jeltsch

1997

European Journal of Biochemistry

321

259

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“…This relaxed specificity was termed star activity. Many REases with star activity have been reported: AvaI ( 14 ), BamHI ( 15–20 ), BanI ( 21 ), Bme126I ( 22 ), BmrI ( 23 ), BspLU111III ( 24 ), BstI ( 25 ), BsuRI ( 26 ), BtsI ( 27 ), CeqI ( 28 ), CviJI ( 29 , 30 ), EcoRI ( 12 , 31–39 ), EcoRV ( 40–42 ), HaeIII ( 14 ), HhaI ( 20 ), HindIII ( 13 , 33 , 43 ), HinfI ( 44 , 45 ), KpnI ( 46 ), MamI ( 47 ), MboII ( 48 ), NcuI ( 49 ), NotI ( 50 ), PpiI ( 51 ), PstI ( 14 , 20 , 52 ), PvuII ( 17 , 43 , 53 , 54 ), RsrI ( 55 , 56 ), SacI ( 43 ), SalI ( 20 ), Sau3AI ( 57 ), SgrAI ( 58 ), SphI ( 59 ), SstI ( 20 ), TaqI ( 60–62 ), Tth111I ( 63 ) and XbaI ( 14 , 20 ). Experimentally, it has been found that the following general conditions may increase star activity: high glycerol concentration (>5% v/v) ( 18–20 , 34 ), high enzyme to DNA ratio (usually >100 U of enzyme per microgram of DNA) ( 12 , 17 , 20 , 34 ), low ionic strength (<25 mM salt) ( 12 , 17 , 20 , 34 , 35 ), high pH (>8.0) ( 41 ), the presence of organic solvents (such as DMSO, ethanol) ( 20 , 34 ,…”

Section: Introductionmentioning

confidence: 99%

The Fidelity Index provides a systematic quantitation of star activity of DNA restriction endonucleases

Hua

Therrien

Blanchard

et al. 2008

Nucleic Acids Research

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Restriction endonucleases are the basic tools of molecular biology. Many restriction endonucleases show relaxed sequence recognition, called star activity, as an inherent property under various digestion conditions including the optimal ones. To quantify this property we propose the concept of the Fidelity Index (FI), which is defined as the ratio of the maximum enzyme amount showing no star activity to the minimum amount needed for complete digestion at the cognate recognition site for any particular restriction endonuclease. Fidelity indices for a large number of restriction endonucleases are reported here. The effects of reaction vessel, reaction volume, incubation mode, substrate differences, reaction time, reaction temperature and additional glycerol, DMSO, ethanol and Mn2+ on the FI are also investigated. The FI provides a practical guideline for the use of restriction endonucleases and defines a fundamental property by which restriction endonucleases can be characterized.

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“…10 hour incubation with a Hinfl concentration of 4 units/Al, 4% glycerol and 100 mM NaCl. Under these conditions very little star activity is observed in assays using human genomic DNA (11).…”

Section: Discussionmentioning

confidence: 99%

“…Secondly, prior knowledge of the recognition sequence of a restriction enzyme is not required for testing its sensitivity to methylation; it is only necessary than the enzyme can cleave the unmethylated substrate at least once. The same PCR procedure can also be used in principle to generate methylated sequences for studies on the effects of methylation on gene regulation in direct DNA transfer experiments (11,12).…”

Section: Discussionmentioning

confidence: 99%

Cytosine methylated DNA synthesized by Taq polymerase used to assay methylation sensitivity of restriction endonucleaseHinfl

Colasanti

Sundaresan

1991

Nucl Acids Res

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We have studied the resistance of cytosine methylated DNA to digestion by the restriction endonuclease HinfI, using a simple PCR procedure to synthesize DNA of known sequence in which every cytosine is methylated at the 5 position. We find that HinfI cannot digest cytosine methylated DNA at the concentrations normally used in restriction digests. Complete digestion is possible using a vast excess of enzyme; under these conditions, the rate of HinfI digestion for cytosine methylated DNA is at least 1440-fold slower than for unmethylated DNA. The presence of an additional methylated cytosine at the degenerate position internal to the recognition sequence does not appear to increase the resistance to HinfI digestion. We also tested HhaII, an isoschizomer of HinfI, and found that it is completely inactive on cytosine methylated DNA. The procedure we have used should be of general applicability in determination of the methylation sensitivities of other restiction enzymes, as well as studies of the effects of methylation on gene expression in direct DNA transfer experiments.

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Digestion conditions resulting in altered cut site specificity forHinfl

Cited by 8 publications

References 1 publication

Recognition and Cleavage of DNA by Type‐II Restriction Endonucleases

Recognition and Cleavage of DNA by Type‐II Restriction Endonucleases

The Fidelity Index provides a systematic quantitation of star activity of DNA restriction endonucleases

Cytosine methylated DNA synthesized by Taq polymerase used to assay methylation sensitivity of restriction endonucleaseHinfl

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