e112 Nucleic Acids Research, 2007, Vol. 35, No. 17 protein extract (e.g. 1 mg). On the other hand, use of a plasmid substrate with the same lesion has involved reaction mixtures with only minimal concentrations of substrate DNA, in view of the diﬃculty of preparing large quantities of plasmid material. This limitation imposes a need for longer incubation periods with higher amounts of protein extract, in order to observe conversion of a signiﬁcant amount of substrate into product. Such substrate-limiting, or eﬃciency-based, conditions can complicate interpretation of reaction kinetics. In addition, there are concerns regarding competing enzymatic activ- ities such as nucleases, as well as plasmid substrate impurities that reduce the eﬀective concentration of substrate. Nevertheless, there can be advantages in using plasmid substrate molecules. For example, artifacts due to protein binding to DNA ends can be eliminated. Also plasmids allow assembly of multi-protein complexes that are too large to be accommodated on oligonucleotide molecules. In addition, plasmids enable use of clamp proteins requiring long and/or circular substrate molec- ules, such as PCNA (23). Finally, there has been a suggestion that plasmid DNA substrates are more relevant to in vivo events than oligonucleotide substrate molecules, however, this latter point has been controversial. Resolution of this debate and uncertainties about choice of substrate DNA can be facilitated by a direct comparison of BER kinetics obtained with each type of DNA bearing the same lesion and sequence context. This comparison has been enabled recently by the introduction of a method for plasmid DNA preparation that yields large amounts of puriﬁed plasmid in a relatively short period (26–30). In the present study, we examined cell extract-mediated uracil-initiated BER using plasmid and oligonucleotide substrates with the U:G mispair in the same sequence context. Repair was measured under steady-state conditions for each type of substrate. Values for Km and Vmax were obtained using laboratory reference extracts from bovine testis and mouse embryonic ﬁbro- blasts (MEF). The results of these analyses indicated that plasmid and oligonucleotide substrates yielded similar Vmax values for uracil-DNA BER activity, although the catalytic eﬃciency (Vmax/Km) of repair with the oligonu- cleotide substrate was slightly higher than with the plasmid substrate. Methods for measuring the ratio of the two BER subpathways, SN BER and LP BER, and for evaluation of the LP BER repair patch size are also discussed. MATERIALS AND METHODS Materials Synthetic oligodeoxyribonucleotides were from Integrated DNA Technologies, Inc. (Coralville, IA, USA) and Oligos Etc. Inc. (Wilsonville, OR, USA). The [a-32P]dCTP and dTTP (3000 Ci/mmol) were from GE HealthCare (Piscataway, NJ, USA). Pfu DNA polymerase was from Stratagene (La Jolla, CA, USA). Endonuclease N.BstNB1, T4 DNA ligase and all other restriction enzymes were from New England Biolabs (Beverly, MA, USA). Plasmids were isolated using Qiagen Plasmid Maxi kits from Qiagen PAGE 2 OF 10 (Valencia, CA, USA). Streptavidin-coated magnetic beads (Dynabeads M-280) were from Invitrogen (Carlsbad, CA, USA). Recombinant human DNA polymerase b (Pol b) was overexpressed and puriﬁed as described previously (31). Human uracil-DNA glycosylase (UDG), apurinic/ apyrimidinic endonuclease (APE) and DNA ligase I were puriﬁed as described previously (32–34). Preparation of lesion-specific plasmid DNA substrates Lesion-speciﬁc BER plasmid substrates were prepared essentially as described previously (29) with slight modiﬁcations. Plasmid pUC19N was derived from pUC19 by inserting a 43-bp phosphorylated oligonucleo- tide (50-GATCGAGTCGAATGCATGCCTCGAGTCT A GAGGTACCAGATCT-30) at the BamHI site position 417. During this process the BamHI site was lost. The resulting pUC19N contained two N.BstNB1 sites ﬂanking 48 bases that could be substituted with a lesion-speciﬁc oligonucleotide. The sequence of the pUC19N plasmid was conﬁrmed by sequencing. A 48-mer oligonucleotide in the sense strand that contained a deﬁned lesion base [uracil or tetrahydrofuran (THF) at position 20, under- scored C above] was replaced between two N.BstNB1 sites as follows: pUC19N (100 mg, 57 pmol) was digested with 500 U of N.BstNB1 at 558C for 2 to 3 h, followed by subsequent incubation with 200 pmol 30-biotin-tagged complementary 48-mer oligonucleotide on a rotary shaker for 30 min at 378C. Then, streptavidin-coated Dynabeads M-280 (400 pmol), pre-equilibrated with 10 mM Tris–HCl, pH 7.5, 0.5 mM EDTA and 1 M NaCl, were added to the reaction mixture, and the mixture was incubated for 2 h at 378C. The 48-bp biotin- tagged DNA, adsorbed onto magnetic beads, was separated from the gapped-pUC19N DNA by placing the reaction mixture tube on a magnet for 2 min to collect the beads. The supernatant was carefully removed with a pipette while the tube remained on the magnet. Gapped- plasmid DNA in the supernatant was extracted twice with phenol/chloroform and precipitated with 95% ethanol. A 20-fold excess of uracil or THF lesion-containing 48-mer oligonucleotide (1.0 nmol) was added to the gapped-pUC19N DNA in 10 mM Tris–HCl, pH 8.0 and 50 mM NaCl. The DNA mixture was annealed at 458C for 4 h, and then incubated at 258C for 15 h with 5000 U of T4 DNA ligase. In order to purify the closed-circular DNA (ccDNA) plasmid from the un-ligated nicked DNA, two rounds of isopycnic centrifugation in CsCl2 with ethidium bromide were performed. The yield of lesion-speciﬁc plasmid DNA was 30–50% of the starting plasmid (i.e. 25 pmol). The purity of each plasmid preparation was evaluated by digestion with XhoI and subsequent electrophoretic separation of ccDNA and linearized DNA. In this assay, the starting plasmid, pUC19N (plasmid without a lesion), was linearized by XhoI digestion, whereas the lesion-speciﬁc substrates were not. This test for contaminating starting plasmid was considered an important precaution as contamination with the starting plasmid was found to be signiﬁcant for some preparations, owing to ineﬃcient removal of 48-mer N.BstNB1 DNA fragment and religation in the last