(denoted in boldface and shown in Figure 3A) and the other overhang being compatible between A and B. The resulting annealed oligonucleotides, Acon, Bcon, Acis and Bcis, were puriﬁed by separation in a 15% native gel followed by electroelution and precipitation. Using T4 DNA ligase, the annealed oligonucleotides were separately ligated to pGEM3zf+ plasmid DNA linearized by EcoRI digestion. Experiments were initially performed with different concen- trations of annealed oligonucleotides and 50-fold molar excess of oligonucleotides to plasmid DNA EcoRI ends was shown to be sufﬁcient for >95% ligation. These four different ligated substrates of DNA (Acon, Bcon, Acis and Bcis) were then further puriﬁed by separation in a 0.6% agarose gel and puri- ﬁcation using the QIAquick gel extraction kit (Qiagen Ltd, Crawley, UK). An initial ligation was performed with T4 DNA ligase in order to show that there was no recircularization, and hence ligation of double-stranded oligonucleotides onto the plasmid backbone had been successful. Analysis of the 32P-ATP end-labelled control and cisplatinated substrate by restriction digest using ApaLI, or HindIII digestion followed by exonuclease III action conﬁrmed the presence of the cis- platin adduct on the manufactured DNA substrate (Figure 3B). NHEJ assay NHEJ extracts were produced essentially as described previ- ously (21,22). End-joining reactions (20 ml) were carried out with 40 mg protein extract from M059K cells and 20 ng DNA substrate in the presence of 50 mM HEPES, pH 8.0, 40 mM KOAc, 1 mM Mg(OAc)2, 1 mM ATP, 1 mM DTT, 0.1 mg/ml BSA and 1 U RNase A at 37 C. For DNA substrates with one cisplatin adduct in the entire molecule, equal amounts (10 ng) of either cisplatinated (Acis and Bcis) or control (Acon and Bcon) substrate were used. For example, 10 ng each of Acis and Bcis substrates were mixed together to provide compatible ends in the reaction mixture (in one reaction) to investigate the effect of a cisplatin adduct on either side of a DSB, while in a reaction with 10 ng each of Acis and Bcon substrates in the reaction mixture the effect of a cisplatin adduct at only one side of the DSB was examined. This was possible because the A substrate alone had incompatible 4 nt overhangs but with the addition of the B substrate, compatible overhangs would be present in the reaction. Protein was removed by incubation with proteinase K (2 mg/ml) and 0.5% (w/v) SDS for 30 min and extraction with Tris-buffered phenol/chloroform/isoamyl alcohol. Each assay was repeated at least three times. Reac- tions were separated on 0.7% agarose gels, and signal quanti- ﬁed by image analysis (Molecular Imager FX and the Quantity One version 4.1.1 software; Bio-Rad Ltd, Hertfordshire, UK), following SYBR Green I staining of the gel. To determine the dependence of DNA joining on DNA-PK or Ku, Wortmannin (10 mM; Sigma, UK) or anti-Ku70 antibody (1:20 dilution; ab87, Abcam, UK), respectively, was included in the reaction mixture in the absence of DNA for 10 min on ice before the addition of DNA and incubation at 37 C. RESULTS NHEJ of cisplatinated plasmid DNA The effect of cisplatin adducts on NHEJ was initially examined using a linear double-stranded 3.2 kb DNA substrate Nucleic Acids Research, 2005, Vol. 33, No. 8 2533 with cohesive 30 four base overhangs produced by PstI diges- tion where the entire DNA molecule had been exposed to a range of cisplatin concentrations. Cisplatination reduced levels of NHEJ by MO59K extract as measured by linear dimer, trimer and higher multimer formation (Figure 1). This effect was clearly dependent on the level of cisplatina- tion achieved and no NHEJ was seen at the highest cisplatin Figure 1. NHEJ of control and cisplatinated DNA substrate. End-joining performed with MO59K extract and DNA substrate treated with cisplatin at the following concentration (nmol) (A) 5.3, (B) 4.4, (C) 3.5, (D) 2.65, (E) 1.1 and (F) buffer only. NHEJ was measured over a time course where DNA was incubated with extract for 0, 0.5, 1, 2, 3 and 4 h (lanes 1–6). The 3.2 kb linear monomer (1·) was joined to form ligated linear dimers (2·), trimers (3·) and higher multimers (m) as indicated. (G) Proportion of DNA substrate ligated against time at the indicated cisplatin concentrations.