Skip Navigation



PEDS Advance Access published online on November 20, 2009
Protein Engineering Design and Selection, doi:10.1093/protein/gzp064
This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
23/2/51    most recent
gzp064v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Takakusagi, K.
Right arrow Articles by Sakaguchi, K.
PubMed
Right arrow PubMed Citation
Right arrow Articles by Takakusagi, K.
Right arrow Articles by Sakaguchi, K.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

© The Author 2009. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

A sulfoglycolipid beta-sulfoquinovosyldiacylglycerol (βSQDG) binds to Met1-Arg95 region of murine DNA polymerase lambda (Mmpol {lambda}) and inhibits its nuclear transit

K. Takakusagi, Y. Takakusagi, K. Ohta, S. Aoki, F. Sugawara and K. Sakaguchi1

Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan

1 To whom correspondence should be addressed. E-mail: kengo{at}rs.noda.tus.ac.jp

Beta-sulfoquinovosyldiacylglycerol (βSQDG) is a synthetic sulfoglycolipid that shows inhibitory activity of DNA polymerase {lambda} (pol {lambda}). Here we identified a βSQDG binding region within murine pol {lambda} (Mmpol {lambda}) using T7 phage display technology. We compared the binding intensity of βSQDG with recombinant phages (phages {lambda}1–6) that displayed different segments of Mmpol {lambda}. The binding assay clearly showed that phage {lambda}1, which displayed the non-structural Met1-Arg95 region including the nuclear localization signal (NLS) and part of the BRCT domain, bound more strongly to βSQDG than the other recombinant phages. Binding assays using recombinant proteins gave similar results, showing specific βSQDG binding to Met1-Arg95 with a KD value of 9.9 nM. Furthermore, in a cell-based assay, nuclear localization of EGFP-pol{lambda} was inhibited in the presence of βSQDG possibly due to binding of βSQDG to NLS. These experiments clearly show that the binding region of βSQDG within Mmpol {lambda} could be successfully identified using T7 phage display technology. We suggest that the strategy we describe here will be of value for identifying the binding site within a protein for small ligands, and will provide information that cannot be obtained using other experimental techniques due to their inherent technical limitations.

Keywords: DNA polymerase lambda/nuclear transit/sulfoquinovosylacylglycerol/T7 phage display

Received July 16, 2009; revised September 5, 2009; accepted October 4, 2009.


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?




Disclaimer: Please note that abstracts for content published before 1996 were created through digital scanning and may therefore not exactly replicate the text of the original print issues. All efforts have been made to ensure accuracy, but the Publisher will not be held responsible for any remaining inaccuracies. If you require any further clarification, please contact our Customer Services Department.