6118 Nucleic Acids Research, 2006, Vol. 34, No. 21 previously (23,37). The wild-type (WT) IN used contains the F185H solubility improving substitution. Protein expression and puriﬁcation was carried out as described previously for this vector (38). IN containing plas- mid constructs were transformed into BL21-CodonPlus(DE3)- RIL E.coli cells (Stratagene), and protein expression was induced with 150 mM isopropyl-b-D-thiogalactopyranoside (IPTG) at an OD600 of 0.5. After 3 h at 37 C, cells were har- vested and pellets were ﬂash frozen in liquid nitrogen and stored at 80 C. Cell pellets from induced cultures were resuspended in lysis buffer [20 mM Tris–HCl (pH 7.9) and 200 mM NaCl] and lysed with 2 mg/ml lysozyme. Proteinase inhibitor cocktail I (1·; Calbiochem) and 10 mM CHAPS (Sigma–Aldrich) were added, and the solution was brought to 1 M NaCl, 5 mM b-mercaptoethanol (b-ME) and 5 mM imidazole. The lysate was then sonicated and cleared by cen- trifugation and passage through a 0.45 mM ﬁlter. The sample was loaded onto a Ni-nitrilotriacetic acid agarose resin (Invitro- gen) that was previously equilibrated with binding buffer [1 M NaCl, 20 mM Tris–HCl (pH 7.9), 5 mM b-ME, 10 mM CHAPS and 10 mM imidazole]. The column was washed with binding buffer and wash buffer [1 M NaCl, 20 mM Tris–HCl (pH 7.0), 5 mM b-ME, 10 mM CHAPS and 25 mM imidazole]. Finally, the column was eluted with elution buffer [1 M NaCl, 20 mM Tris–HCl, 5 mM b-ME, 10 mM CHAPS and 0.2 M imidazole], and buffer exchange was carried out using a YM-30 Centriprep (Millipore) to transfer the protein into IN storage buffer [ISB; 20% glycerol, 727 mM NaCl, 7.27 mM HEPES (pH 7.5), 7.27 mM b-ME and 7.27 mM ZnSO4]. Oligonucleotides All oligonucleotides were purchased from Integrated DNA Technologies. The PCR-boosted disintegration substrate (Figure 1B, top) consists of four oligonucleotides, U5CA-20 d(ACTGCTAGAGATTTTCCACA), U5T-55 d(TGTGGAA AATCTCTAGCACATTCCTTATCCCACTAGCTCTCTCG- ACGCAGGACTC), TTop-60 d(GTGTGCCCGTCTGTTGT- GTCTCTCAGTGGCGCCCGAACAGGGACACATTACTT- ATCTAC) and Tbottom-32 DNA d(AGTGGGATAAGGAA- TGGTAGATAAGTAATGTG) or Tbottom-32 RNA r(AGU- GGGAUAAGGAAUGGUAGAUAAGUAAUGUG). As seen in Figure 1B, the substrate encodes the viral U5 terminus (LTR end) and a region representing target DNA (target). Primers for product quantiﬁcation are Dis F d(TGTGT- GCCCGTCTGTTGTGT), Dis R d(GAGTCCTGCGTCGA- GAGAGC), and the TaqMan Probe, Dis Probe [50-FAM- d(CAGTGGCGCCCGAACAGGGA)-TAMNph-30]. A 97mer oligonucleotide, Dis Full-97 d(TGTGTGCCCGTCTGTTGT- GTCTCTCAGTGGCGCCCGAACAGGGACACATTACTT- ATCTACCATTCCTTATCCCACTAGCTCTCTCGACGCA- GGACTC), representing the expected disintegration product was used to generate a standard curve. A ‘reverse polarity’ substrate (Figure 4A) was also con- structed using Tbottom-32 RNA (see above), U5–18 d(TGTG- GAAAATCTCTAGCA), Ttop-37 d(CATTCCTTATCCCA- CTAGCTCTCTCGACGCAGGACTC), and TU5–78 d(TGT- GTGCCCGTCTGTTGTGTCTCTCAGTGGCGCCCGAAC- AGGGACACATTACTTATCTACTGCTAGAGATTTTCC- ACA). Primers for product quantiﬁcation are the same as those used for the standard substrate (Figure 1B, upper). Substrates were made by mixing the constituent oligonu- cleotides at a concentration of 800 nM each in water (test reactions were performed with substrates annealed in 0.1 M NaCl and results were unchanged) and slow cooling from 95 C (all DNA substrates) or 65 C (RNA containing sub- strates) down to 4 C at a rate of 0.5 C per 1.5 min. For reac- tion containing radiolabeled substrate TTop-60 (see above) was 50 end-labeled with [g-32P]ATP using polynucleotide kinase. The radiolabeled oligonucleotide was then annealed to the other oligonucleotides as above. The annealed substrate was applied to a G50-microspin column to remove unincor- porated [g-32P]ATP. For reactions with radiolabeled PCR primer, Dis F was 50 end-labeled with [g-32P]ATP using poly- nucleotide kinase and was then applied to a G50-microspin column to remove unincorporated [g-32P]ATP. qPCR-based disintegration assay Unless otherwise noted, reactions were carried out in 40 ml total volume with 30 nM substrate, 19.8 mM HEPES (pH 7.3), 1.8 mM b-ME, 10 mM MnCl2 or 20 mM MgCl2, and 1 mM IN. Reactions were started by addition of 2 ml 20 mM IN (diluted in ISB). Reactions were incubated at 37 C for the indicated length of time and stopped by addition of 5 ml of a solution of 4.5% SDS and 1.35 mg/ml Proteinase K (Roche Applied Science) and incubation at 37 C for an additional 45 min. Reactions were heated at 65 C for 5 min prior to puriﬁcation via the QIAquick Nucleotide Removal Kit (Qiagen). DNA was eluted in the provided Buffer EB. Reactions were quantiﬁed by real-time qPCR after dilution in Buffer EB to obtain DNA samples in the linear range. qPCR were carried out in 25 ml total volumes on an ABI Prism 7700 (Applied Biosystems) using the TaqMan Uni- versal PCR Master Mix (Applied Biosystems), 300 nM each primer, 200 nM Late-RT-Probe. A total of 5 ml of reac- tion sample or standard curve were added per reaction. The universal cycling conditions (50 C 2 min, 95 C 10 min, 40 cycles of 95 C 15 s and 60 C 1 min) recommended by Applied Biosystems were used. Comparative reactions with 32P-labeled substrate followed the same protocol for the transesteriﬁcation portion of the qPCR-based disintegration assay and were stopped by addi- tion of 200 ml formamide loading buffer [80% (w/v) deion- ized formamide, 10 mM EDTA (pH 8.0), 1 mg/ml xylene cyanol FF and 1 mg/ml bromophenol blue] rather than by SDS/Proteinase K treatment and nucleotide removal kit. A total of 5 ml of each reaction was applied to an 8% denaturing-polyacrylamide gel and analyzed using OptiQuant software on a Storm PhosphorImager (GE Healthcare). Reactions quantiﬁed using 32P-labeled Dis F primer were performed on 1000-fold dilutions of nucleotide removal kit puriﬁed transesteriﬁcation reactions using the same condi- tions as for the qPCR analysis (no probe was added). PCR products were diluted 5-fold in formamide loading buffer (see above) and 5 ml of each sample was analyzed by 8% denaturing-polyacrylamide gel as discussed above. qPCR-based DNA transesterification reactions with metal only These reactions, carried out in the absence of IN, followed the protocol discussed above. Reactions were carried out in