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Nucleic Acids Research Advance Access published online on August 24, 2007
Nucleic Acids Research, doi:10.1093/nar/gkm639
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© 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. 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.


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