Solution parameters modulating DNA binding specificity of the restriction endonuclease EcoRV
نویسندگان
چکیده
منابع مشابه
Fluorescent substrates for the EcoRV restriction endonuclease.
In the presence of Mg2+, the EcoRV restriction endonuclease cleaves DNA specifically at the sequence GAT'ATC (where denotes the point of scission) [ I ] . Even the change of a single base pair within this sequence will lead to a million fold reduction of EcoRV activity [2]. Paradoxically, gel retardation experiments in the absence of Mg2+ show that, unlike EcoRl and many other type 11 restricti...
متن کاملCrosslinking the EcoRV restriction endonuclease across the DNA-binding site reveals transient intermediates and conformational changes of the enzyme during DNA binding and catalytic turnover.
EcoRV completely encircles bound DNA with two loops, forming the entry and exit gate for the DNA substrate. These loops were crosslinked generating CL-EcoRV which binds and releases linear DNA only slowly, because threading linear DNA into and out of the DNA-binding 'tunnel' of CL-EcoRV is not very effective. If the crosslinking reaction is carried out with a circular bound DNA, CL-EcoRV is hyp...
متن کاملRational engineering of type II restriction endonuclease DNA binding and cleavage specificity
The type II restriction endonucleases are indispensible tools for molecular biology. Although enzymes recognizing nearly 300 unique sequences are known, the ability to engineer enzymes to recognize any sequence of choice would be valuable. However, previous attempts to engineer new recognition specificity have met limited success. Here we report the rational engineering of multiple new type II ...
متن کاملCatalytic and DNA binding properties of PvuII restriction endonuclease mutants.
The role of particular residues of the PvuII endonuclease in DNA binding and cleavage was studied by mutational analysis using a number of in vivo and in vitro approaches. While confirming the importance of residues predicted to be involved directly in function by the crystal structure, the analysis led to several striking results. Aspartate 34, which contacts the central base pair of the PvuII...
متن کاملNucleotide flips determine the specificity of the Ecl18kI restriction endonuclease.
Restricion endonuclease Ecl18kI is specific for the sequence /CCNGG and cleaves it before the outer C to generate 5 nt 5'-overhangs. It has been suggested that Ecl18kI is evolutionarily related to NgoMIV, a 6-bp cutter that cleaves the sequence G/CCGGC and leaves 4 nt 5'-overhangs. Here, we report the crystal structure of the Ecl18kI-DNA complex at 1.7 A resolution and compare it with the known...
متن کاملذخیره در منابع من
با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید
ژورنال
عنوان ژورنال: FEBS Journal
سال: 2011
ISSN: 1742-464X
DOI: 10.1111/j.1742-4658.2011.08198.x