Skip Navigation


Nucleic Acids Research Advance Access originally published online on August 24, 2007
Nucleic Acids Research 2007 35(17):e112; doi:10.1093/nar/gkm639
This Article
Right arrow Full Text Freely available
Right arrow Print PDF (2732K) Freely available
Right arrow Screen PDF (444K) Freely available
Right arrowOA All Versions of this Article:
35/17/e112    most recent
gkm639v1
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 arrow Commercial Re-use Guidelines
for Open Access NAR Content
Google Scholar
Right arrow Articles by Hou, E. W.
Right arrow Articles by Wilson, S. H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hou, E. W.
Right arrow Articles by Wilson, S. H.
Related Collections
Right arrow Repair
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Nucleic Acids Research, 2007, Vol. 35, No. 17 e112
© 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Methods Online

Comparative assessment of plasmid and oligonucleotide DNA substrates in measurement of in vitro base excision repair activity

Esther W. Hou, Rajendra Prasad, Kenjiro Asagoshi, Aya Masaoka and Samuel H. Wilson*

Laboratory of Structural Biology, National Institute of Environmental Health Sciences, National Institutes of Health, 101 T.W. Alexander Drive, Research Triangle Park, NC 27709, USA

*To whom correspondence should be addressed: Tel: 919 541 3267; Fax: 919 541 3592; Email: wilson5{at}niehs.nih.gov

Received June 22, 2007. Revised August 1, 2007. Accepted August 1, 2007.

Mammalian base excision repair (BER) is mediated through at least two subpathways designated ‘single-nucleotide’ (SN) and ‘long-patch’ (LP) BER (2-nucleotides long/more repair patch). Two forms of DNA substrate are generally used for in vitro BER assays: oligonucleotide- and plasmid-based. For plasmid-based BER assays, the availability of large quantities of substrate DNA with a specific lesion remains the limiting factor. Using sequence-specific endonucleases that cleave only one strand of DNA on a double-stranded DNA substrate, we prepared large quantities of plasmid DNA with a specific lesion. We compared the kinetic features of BER using plasmid and oligonucleotide substrates containing the same lesion and strategic restriction sites around the lesion. The Km for plasmid DNA substrate was slightly higher than that for the oligonucleotide substrate, while the Vmax of BER product formation for the plasmid and oligonucleotide substrates was similar. The catalytic efficiency of BER with the oligonucleotide substrate was slightly higher than that with the plasmid substrate. We conclude that there were no significant differences in the catalytic efficiency of in vitro BER measured with plasmid and oligonucleotide substrates. Analysis of the ratio of SN BER to LP BER was addressed using cellular extracts and a novel plasmid substrate.


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.