3OOR

I-SceI mutant (K86R/G100T)complexed with C/G+4 DNA substrate

Summary for 3OOR

• Entry DOI: 10.2210/pdb3oor/pdb
• Related: 1R7M 3C0W 3C0X 3OOL
• Descriptor: Intron encoded endonuclease I-SceI, 5'-D(*CP*AP*CP*GP*CP*TP*AP*GP*GP*GP*AP*TP*AP*AP*CP*CP*GP*GP*GP*TP*AP*AP*TP*AP*C)-3', 5'-D(*GP*GP*TP*AP*TP*TP*AP*CP*CP*CP*GP*GP*TP*TP*AP*TP*CP*CP*CP*TP*AP*GP*CP*GP*T)-3', ... (5 entities in total)
• Functional Keywords: homing endonuclease, intron homing, laglidadg, hydrolase-dna complex, i-scei mutant (k86r/g100t), hydrolase/dna
• Biological source: Saccharomyces cerevisiae (yeast)
• Cellular location: Mitochondrion: P03882
• Total number of polymer chains: 3
• Total formula weight: 43536.78

Authors

Joshi, R.,Chen, J.-H.,Golden, B.L.,Gimble, F.S. (deposition date: 2010-08-31, release date: 2010-11-17, Last modification date: 2023-09-06)

Primary citation

Joshi, R.,Ho, K.K.,Tenney, K.,Chen, J.H.,Golden, B.L.,Gimble, F.S.
Evolution of I-SceI Homing Endonucleases with Increased DNA Recognition Site Specificity.
J.Mol.Biol., 405:185-200, 2011

PubMed Abstract: 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: 21029741
DOI: 10.1016/j.jmb.2010.10.029

Experimental method

X-RAY DIFFRACTION (2.5 Å)