EcoRI★ activity: Enzyme modification or activation of accompanying endonuclease?

Tikchonenko, T.I.; Карамов, Э. В.; Zavizion, Boris; Naroditsky, Boris S.

doi:10.1016/0378-1119(78)90018-5

Cited by 41 publications

(17 citation statements)

References 14 publications

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“…This ‘star activity’ (EcoRI*) was also observed in the presence of organic solvents, such as glycerol or DMSO (71–73), and when Mg 2+ is replaced by Mn 2+ (74). Co 2+ and Zn 2+ also support DNA cleavage, but unlike Mn 2+ do not result in star activity (75).…”

Section: Introductionmentioning

confidence: 81%

Type II restriction endonucleases—a historical perspective and more

2014

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This article continues the series of Surveys and Summaries on restriction endonucleases (REases) begun this year in Nucleic Acids Research. Here we discuss ‘Type II’ REases, the kind used for DNA analysis and cloning. We focus on their biochemistry: what they are, what they do, and how they do it. Type II REases are produced by prokaryotes to combat bacteriophages. With extreme accuracy, each recognizes a particular sequence in double-stranded DNA and cleaves at a fixed position within or nearby. The discoveries of these enzymes in the 1970s, and of the uses to which they could be put, have since impacted every corner of the life sciences. They became the enabling tools of molecular biology, genetics and biotechnology, and made analysis at the most fundamental levels routine. Hundreds of different REases have been discovered and are available commercially. Their genes have been cloned, sequenced and overexpressed. Most have been characterized to some extent, but few have been studied in depth. Here, we describe the original discoveries in this field, and the properties of the first Type II REases investigated. We discuss the mechanisms of sequence recognition and catalysis, and the varied oligomeric modes in which Type II REases act. We describe the surprising heterogeneity revealed by comparisons of their sequences and structures.

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Section: Introductionmentioning

confidence: 81%

Type II restriction endonucleases—a historical perspective and more

2014

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“…For EcoRI, intersecting spheres means confusion of sequences. For example instead of only binding GAATTC, thermal noise could flexibly distort the EcoRI protein so that it might also bind AAATTC, leading to inappropriate digestion of the genome [`star activity' (64)]. The coding space must map to the physical molecule, but the mapping can be different in different cases, just as an IF statement in a computer language may be supported by relays, vacuum tubes, transistors, or proteins and DNA in a genetic control circuit (65).…”

Section: Resultsmentioning

confidence: 99%

70% efficiency of bistate molecular machines explained by information theory, high dimensional geometry and evolutionary convergence

Schneider¹

2010

Nucleic Acids Research

101

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The relationship between information and energy is key to understanding biological systems. We can display the information in DNA sequences specifically bound by proteins by using sequence logos, and we can measure the corresponding binding energy. These can be compared by noting that one of the forms of the second law of thermodynamics defines the minimum energy dissipation required to gain one bit of information. Under the isothermal conditions that molecular machines function this is joules per bit ( is Boltzmann's constant and T is the absolute temperature). Then an efficiency of binding can be computed by dividing the information in a logo by the free energy of binding after it has been converted to bits. The isothermal efficiencies of not only genetic control systems, but also visual pigments are near 70%. From information and coding theory, the theoretical efficiency limit for bistate molecular machines is ln 2 = 0.6931. Evolutionary convergence to maximum efficiency is limited by the constraint that molecular states must be distinct from each other. The result indicates that natural molecular machines operate close to their information processing maximum (the channel capacity), and implies that nanotechnology can attain this goal.

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“…We hypothesized that use of DMSO and glycerol might change target site preference and increase transposition activity based on the fact that these molecules can modify water structure around proteins in aqueous solution resulting in increased or decreased enzyme activity [Pasha et al, 2002;Rehan and Younus, 2006;Wiggers et al, 2007]. The use of DMSO or glycerol can change the DNA-binding specificity of some restriction enzymes [Bishop, 1979;Conlan et al, 1999;Izsvák and Duda, 1989;Tikchonenko et al, 1978]. Although 15% (v/v) glycerol and/or 15% (v/v) DMSO have been reported to decrease the requirement for Mu enhancer and other accessory proteins for transposition [Clubb et al, 1996;Mizuuchi and Mizuuchi, 1989], it has not been previously shown that the presence of these components in the buffer could result in more active transposition with decreased target site preference.…”

Section: Discussionmentioning

confidence: 99%

N-Terminal Domain-Deleted Mu Transposase Exhibits Increased Transposition Activity with Low Target Site Preference in Modified Buffers

Kim¹,

Morrison²

2008

Microb Physiol

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Background: Transposition using MuA and the Mu-transposon is frequently used for insertional and deletional mutagenesis and to introduce primer sequences into DNA of unknown sequence. However, mutagenesis studies are often limited by the number of transposition events taking place within a small target sequence. Although the intrinsic target site preference of MuA is low, it nevertheless may limit the recovery of desired events. Methods: In the current study, we have compared the transposition activity of wild-type, C-terminally deleted, N-terminally deleted and both C- and N-terminally deleted MuA. Results: We found that N-terminally deleted MuA was most active with an about sevenfold increase in transposition efficiency compared to wild-type. In addition, we identified a novel reaction condition in which the transposition activity of N-terminally deleted MuA was increased about 45-fold. Conclusion: The use of the N-terminally deleted MuA with the newly established condition enhanced the relative transposition activity about 280-fold compared to that of wild-type MuA with the conventional buffer. In addition, the optimized buffer decreased the target site preference of N-terminally deleted MuA resulting in a more even distribution of integration events throughout the target gene.

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EcoRI★ activity: Enzyme modification or activation of accompanying endonuclease?

Cited by 41 publications

References 14 publications

Type II restriction endonucleases—a historical perspective and more

Type II restriction endonucleases—a historical perspective and more

70% efficiency of bistate molecular machines explained by information theory, high dimensional geometry and evolutionary convergence

N-Terminal Domain-Deleted Mu Transposase Exhibits Increased Transposition Activity with Low Target Site Preference in Modified Buffers

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