PAGE 9 OF 10 Nucleic Acids Research, 2007, Vol. 35, No. 17 e112 Figure 7. Restriction analysis and quantiﬁcation of dCMP incorporation into the repair patch produced during LP BER in MEF extract. The repair reaction was performed with THF-containing plasmid, pUN2 and MEF extract. (A) BER reaction was performed in a 10 ml reaction mixture that contained 20 nM pUN2, 10 mg MEF extract, and 32P-dCTP. Incubation was for 30 min at 378C. The reaction products were analyzed as in Figure 3. The restriction enzymes used are shown at the top of the PhosphorImager panel. The positions of the restricted DNA fragments are indicated on the left-hand side of the image. (B) DNA fragment sizes and description of DNA synthesis products are indicated. Sites of dCMP incorporation (ﬁlled circle) in the repair patch are indicated. (C) The incorporation of dCMP (%) in diﬀerent BER fragments was plotted in a bar diagram. Conversion of arbitrary PhosphorImager units into dCMP incorporation was performed as in Figure 6. The experiments were repeated three times, and the PhosphorImage of a representative experiment is shown. extract-based repair synthesis, the Km diﬀerence between the two forms of DNA may need to be taken into account. In addition to characterizing rates of BER as a function of substrate form, we examined methods for analysis of repair patch length in the LP BER subpathway. Our initial experiments in this eﬀort made use of phosphorothioate dNMP incorporation into the repair patch and subsequent exonuclease III digestion of products [data not shown, and ref. (37)]. This approach yielded spurious results (data not shown). The main diﬃculty stemmed from the capacity of exonuclease III to digest into the repair patches containing phosphorothioate dNMPs, leading to an underestimate of repair patch length. On the other hand, attempts to reduce the digestion strategically (time or enzyme concentration) generally lead to an over- estimate of repair patch length. Therefore, we concluded that reliable measurements of repair patch length could not be obtained with this approach. The approach described here, however, appeared to be reliable and was limited only by the technical requirements to obtain pure substrate preparations, restriction digestion of product molecules to completion, and attention to accurate quantiﬁcation of the amount of labeled dNMP incorporated. ACKNOWLEDGEMENTS This research was supported by the Intramural Research Program of the NIH, National Institute of Environmental Health Sciences. We thank, Dr. William A. Beard and Dr. Julie K. Horton, NIEHS/NIH, for critical reading of this manuscript. We also thank Jennifer P. Myers for editorial assistance. Funding to pay the open access publication charges for this article was also provided by the NIEHS, NIH. Conﬂict of interest statement. None declared. REFERENCES 1. Klungland,A., Rosewell,I., Hollenbach,S., Larsen,E., Daly,G., Epe,B., Seeberg,E., Lindahl,T. and Barnes,D.E. (1999)