Published online 24 August 2007 Nucleic Acids Research, 2007, Vol. 35, No. 17 e112 doi:10.1093/nar/gkm639 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 Received June 22, 2007; Revised and Accepted August 1, 2007 ABSTRACT 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 con- taining 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 oligonucleo- tide 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 differ- ences in the catalytic efficiency of in vitro BER measured with plasmid and oligonucleotide sub- strates. Analysis of the ratio of SN BER to LP BER was addressed using cellular extracts and a novel plasmid substrate. INTRODUCTION To understand cellular responses to endogenous genotoxic stress, it is important to have insight into the DNA repair process known as base excision repair (BER). BER is involved in repairing base lesions and single-strand breaks that occur thousands of times during the average mammalian cell cycle (1–3) and is generally considered to involve two subpathways deﬁned by the size of the repair patch and the enzymes involved (4–7). These subpathways are termed single-nucleotide (SN) BER and long-patch (LP) BER. As these names imply, in SN BER one nucleotide in the damaged strand is excised and replaced (8–11), whereas LP BER excises and replaces several nucleotides in the damaged strand (6,9). LP BER appears to be a backup subpathway in cases where the SN BER system stalls or where the DNA lesion is refractory to the enzymatic steps of the SN BER subpathway (6,9,12–14). Components of the BER system are constitutively expressed in mammalian cells, but also exhibit widely divergent tissue-speciﬁc expression levels plus up- or down-regulation after genotoxic stress and cytokine exposure (15–20). This dynamic regulatory picture sug- gests the potential for cell type speciﬁc diﬀerences in cellular capacity for BER and also for diﬀerences in relative use of the two BER subpathways, since there are distinct enzymes and accessory factors involved in each (4–7,10,21–25). Methods for characterization of the over- all amount and eﬃciency of BER in vivo are limited, and improved methods for quantitative measurement of repair capacity and subpathway choice are needed for studies of extract-mediated BER. In recent years, research in the BER area has been greatly facilitated through measurements of in vitro BER activity using oligonucleotide and plasmid substrates containing a lesion in a deﬁned site and sequence context (4–6,8,10,26). There has been debate, however, on advantages and disadvantages regarding the form of DNA used as substrate, i.e. plasmid versus oligonucleo- tide. For example, oligonucleotide substrates oﬀer the advantage of relative ease of preparation of large amounts of pure material, and hence the option of preparing in vitro reaction mixtures with an excess of DNA substrate. Under this condition, steady-state measure- ments of repair activity of extracts can be obtained using short incubation periods (e.g. 1 min) and small amounts of *To whom correspondence should be addressed. Tel: 919 541 3267; Fax: 919 541 3592; Email: wilson5@niehs.nih.gov 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.