2532 Nucleic Acids Research, 2005, Vol. 33, No. 8 repair pathway, the latter being more error prone. The HR pathway requires a DNA template strand, usually a sister chromatid, whereas the NHEJ pathway has no such require- ment [reviewed in (7–9)]. The basic NHEJ pathway [reviewed in (7–10)] requires a number of proteins. Initially, the heterodi- mer Ku70/Ku80 recognizes and binds to the free DNA ends at a DSB in a sequence-independent manner. DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is then recruited to the Ku heterodimer to form DNA-PK holoenzyme. The Ku heterodimer can be translocated along the DNA strand as the DNA passes through the hole in the Ku heterodimer ring-like shape (11). The DNA-PK holoenzyme has kinase activity, which recruits and co-ordinates repair proteins that may be important in phosphorylating proteins in this pathway. Finally, XRCC4 stimulates DNA ligase IV to join the broken ends. Other components thought to be involved depending on the type of DNA ends to be joined are inositol hexakis- phosphate (12), Artemis (13), MRN complex (14) and DNA polymerase m (15). Previous work has shown that the presence of a cisplatin adduct near a DSB may decrease the rate of Ku translocation along the DNA strand and lowers the DNA-PK kinase activity (16–18). However, these investigations were performed in the presence of high levels of the puriﬁed components Ku70/80, so that multiple Ku heterodimer molecules were shown to bind and translocate along the DNA used. In addition, only the initial steps of the NHEJ pathway were examined, so that the ability to ligate two ends of DNA was not assessed. These results have been conﬁrmed more recently (19) showing that the cisplatin adducts do not alter the ability of Ku to bind DNA ends, but do impair the translocation of Ku complex along the DNA. Using a ligation assay with recombinant Ku, XRCC4 and DNA ligase IV, the cisplatin adducts were able to dramatically inhibit DNA ligation, whereas if no Ku was present the high ligation levels were very slightly decreased in the cisplatinated DNA. The ability to perform ligation was dependent on the DNA substrate length. In the present work, we examined the effect of cisplatin adduct formation on the ability to perform NHEJ using cell-free extracts, which contain the full complement of factors required for NHEJ, rather than recombinant proteins. We also used large 3.2 kb DNA substrates rather than smaller DNA frag- ments, which may limit the ability to form appropriate DNA repair complexes around the DSB. DNA substrates with vary- ing degrees of cisplatination were used ranging from sub- strates where the entire DNA was cisplatinated, and hence containing many different adduct types, to substrates with a single intrastrand cis-[Pt(NH3)2fd(GpTpG)-N7(1),-N7(3)g] [abbreviated as 1,3-d(GpTpG)] cisplatin adduct 10 bases from the end of the DSB. We show that substrates containing single, deﬁned terminal cisplatin adducts are more informative than ‘globally’ cisplatinated substrates containing both terminal and internal adducts in determining the effect of cisplatin on NHEJ. MATERIALS AND METHODS Production of DNA substrates Cisplatination of the linear 3.2 kb pGEM3zf+ plasmid DNA (Promega, Southampton, UK) was carried out by the incubation of 1 mg of the PstI restriction enzyme cut DNA with 1.1–5.3 nmol of cisplatin (Sigma, Gillingham, UK) in 3 mM NaCl, 0.5 mM Na2HPO4 and 0.5 mM NaH2PO4 at 37 C over- night. Mock-treated DNA was incubated in buffer alone. The reaction was stopped by the addition of 0.5 M NaCl, and the DNA was puriﬁed using G25 spin columns (Amersham Phar- macia, Buckinghamshire, UK). The sequence surrounding the PstI site (shown in boldface) and the space with a underline denotes the position of the restriction: 50-ACTATAGAA- TACTCAAGCTTGCATGCCTGCA_GGTCGACACTAGT- GGATCCAAAGAATTCAAAAAGCTTCT-30. Cisplatination of plasmid DNA was also carried out on the substrates shortcon and shortcis, which were constructed by ligating into the EcoRI–HindIII site of pGEM3zf+ the following annealed nucleotides, respectively: 50-AAT- TCATTATAATATTTATC_AATTGATATAAATTATA-30 dimerized with 50-AGCTTATAATTTATATC_AATTGAT- AAATATTATATG-30 and 50-AATTCATTGTGATATTTAT- C_AATTGATATAAATCACA-30 dimerized with 50-AGC- TTGTGATTTATATC_AATTGATAAATATCACAATG-30. The region contains the MfeI restriction site (shown in bold- face), which was used to linearize the substrates for end-joining reactions, and the space with a underline denotes the position of the restriction. The shortcon and shortcis differ only in that a GTG sequence is present in the shortcis substrate as shown in italics. The longer substrates (longcon and longcis) contained the following sequence ligated into the EcoRI–HindIII site of pGEM3zf+. The longcon sequence was 50-AATTCAATA- TAATTTTATAATATTATAATATTTATC_AATTGATAT- AAATTATAATTTATAATTATTTATATTA-30 and the long- cis sequence was 50-AATTCAATATAATTTTATAATATT- GTGATATTTATC_AATTGATATAAATCACAATTTATA- ATTATTTATATTA-30. The longcon and longcis differ only in that a GTG sequence is present in the longcis substrate as shown in italics. DNA substrates with one cisplatin adduct in the entire molecule were produced using a 24 base oligonucleotide containing the GTG site for cisplatination shown in boldface (50-TCTTCTTCTGTGCACTCTTCTTCT-30). This 24mer was puriﬁed by 15% denaturing PAGE (Sequagel, National Diagnostics, Hessle, UK), identiﬁcation by staining with SYBR Green I (Molecular Probes, Leiden, The Netherlands), and isolated by electroelution (Elutrap, Schleicher and Schuell, London, UK) followed by ethanol precipitation. Cisplatination was carried out as described previously and uncisplatinated and cisplatinated oligonucleotides were separ- ated on a 15% denaturing polyacrylamide gel as described previously (20). The cisplatinated and uncisplatinated DNA were identiﬁed by UV shadowing and electroeluted. The con- trol (con) or cisplatinated (cis) 24mer were annealed to either oligonucleotide A (50-P-AATTAGAAGAAGAGTGCACA- GAAGAAGACGCC-30) or B (50-P-AATTAGAAGAAGAG- TGCACAGAAGAAGAGGCG-30) by heating equimolar amounts of each oligonucleotide at 60 C for 5 min and leaving to cool slowly over 2 h at room temperature. Oligonucleotides A and B are identical in sequence apart from 4 nt shown above in italics. Both oligonucleotides A and B were phosphorylated at the 50 end and were designed such that annealing of either with the 24mer (either con or cis) produced a double-stranded molecule containing a 4 nt overhang at each end, with one overhang being compatible with EcoRI digested plasmid DNA