Home | My Profile | Contact Us
Research Trends Products  |   order gateway  |   author gateway  |   editor gateway  
ID:
Password:
Register | Forgot Password

Author Resources
 Author Gateway
 Article submission guidelines

Editor Resources
 Editor/Referee Gateway

Agents/Distributors
 Regional Subscription Agents/Distributors
 
Current Topics in Biochemical Research   Volumes    Volume 16 
Abstract
Evolutionary adaptation of the GIY-YIG domain for specific DNA binding in I-CreII
Weihua Qiu, Youzhong Guo, David L. Herrin
Pages: 17 - 28
Number of pages: 12
Current Topics in Biochemical Research
Volume 16 

Copyright © 2014 Research Trends. All rights reserved

ABSTRACT
 
Homing endonucleases promote gene invasion by cleaving specific DNA targets, and they typically contain one of several conserved catalytic domains, plus additional motifs that provide specific DNA binding. However, at least two enzymes from green algal chloroplasts, I-CmoeI and I-CreII, have two apparent catalytic domains (H-N-H and GIY-YIG, respectively), and lack most conserved DNA-binding motifs. Mutagenesis of I-CreII indicated that the H-N-H motif is catalytic, and, based on DNA-affinity measurements, that the GIY-YIG motif could be involved in DNA-binding. Here we have used footprinting techniques to investigate target DNA binding by native and substituted forms of I-CreII. Hydroxyl radical footprinting with wild-type I-CreII indicated that the tightest binding occurs downstream of the cleavage site, though binding at the cleavage site was apparent on the top strand, which is the first one cleaved by I-CreII. DNase I footprinting was possible with the variants that have reduced DNA affinity, as well as wild-type. The GIY-YIG variant, G231E/K245A, showed a strong differential effect within the footprint, especially with the hyper-cleavage of several nt just downstream of the cleavage site on the top strand. Structural modeling of the GIY-YIG domain using the solved I-TevI structure as a template indicated that I-CreII has a β-hairpin insertion that interrupts an α-helix normally required for catalysis. These results indicate that the GIY-YIG domain in I-CreII plays an important, if not dominant, role in specific DNA binding. Moreover, they suggest that the evolution of the GIY-YIG domain into this role may have begun with the β-hairpin insertion, which inactivated its catalytic ability.
View Full Article  


 
search


E-Commerce
Buy this article
Buy this volume
Subscribe to this title
Shopping Cart

Quick Links
Login
Search Products
Browse in Alphabetical Order : Journals
Series/Books
Browse by Subject Classification : Journals
Series/Books

Miscellaneous
Ordering Information Ordering Information
Downloadable forms Downloadable Forms