Linked Life Data

21029741

Source:http://linkedlifedata.com/resource/pubmed/id/21029741

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
1
pubmed:dateCreated
2010-12-21
pubmed:abstractText
Elucidating how homing endonucleases undergo changes in recognition site specificity will facilitate efforts to engineer proteins for gene therapy applications. I-SceI is a monomeric homing endonuclease that recognizes and cleaves within an 18-bp target. It tolerates limited degeneracy in its target sequence, including substitution of a C:G(+4) base pair for the wild-type A:T(+4) base pair. Libraries encoding randomized amino acids at I-SceI residue positions that contact or are proximal to A:T(+4) were used in conjunction with a bacterial one-hybrid system to select I-SceI derivatives that bind to recognition sites containing either the A:T(+4) or the C:G(+4) base pairs. As expected, isolates encoding wild-type residues at the randomized positions were selected using either target sequence. All I-SceI proteins isolated using the C:G(+4) recognition site included small side-chain substitutions at G100 and either contained (K86R/G100T, K86R/G100S and K86R/G100C) or lacked (G100A, G100T) a K86R substitution. Interestingly, the binding affinities of the selected variants for the wild-type A:T(+4) target are 4- to 11-fold lower than that of wild-type I-SceI, whereas those for the C:G(+4) target are similar. The increased specificity of the mutant proteins is also evident in binding experiments in vivo. These differences in binding affinities account for the observed ?36-fold difference in target preference between the K86R/G100T and wild-type proteins in DNA cleavage assays. An X-ray crystal structure of the K86R/G100T mutant protein bound to a DNA duplex containing the C:G(+4) substitution suggests how sequence specificity of a homing enzyme can increase. This biochemical and structural analysis defines one pathway by which site specificity is augmented for a homing endonuclease.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Jan
pubmed:issn
1089-8638
pubmed:author
pubmed:copyrightInfo
Copyright © 2010 Elsevier Ltd. All rights reserved.
pubmed:issnType
Electronic
pubmed:day
7
pubmed:volume
405
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
185-200
pubmed:dateRevised
2011-10-6
pubmed:meshHeading
pubmed-meshheading:21029741-Amino Acid Sequence, pubmed-meshheading:21029741-Amino Acid Substitution, pubmed-meshheading:21029741-Crystallography, X-Ray, pubmed-meshheading:21029741-DNA, pubmed-meshheading:21029741-Deoxyribonucleases, Type II Site-Specific, pubmed-meshheading:21029741-Directed Molecular Evolution, pubmed-meshheading:21029741-Models, Molecular, pubmed-meshheading:21029741-Molecular Sequence Data, pubmed-meshheading:21029741-Mutant Proteins, pubmed-meshheading:21029741-Protein Binding, pubmed-meshheading:21029741-Protein Structure, Tertiary, pubmed-meshheading:21029741-Proton-Translocating ATPases, pubmed-meshheading:21029741-Saccharomyces cerevisiae, pubmed-meshheading:21029741-Saccharomyces cerevisiae Proteins, pubmed-meshheading:21029741-Sequence Alignment, pubmed-meshheading:21029741-Substrate Specificity
pubmed:year
2011
pubmed:articleTitle
Evolution of I-SceI homing endonucleases with increased DNA recognition site specificity.
pubmed:affiliation
Department of Biochemistry, Purdue University, West Lafayette, IN 47907, USA.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, Non-P.H.S., Research Support, N.I.H., Extramural