2536 Nucleic Acids Research, 2005, Vol. 33, No. 8 Figure 3. Manufacture of substrate with a single 1,3-d(GpTpG) cisplatin adduct. (A) Diagrammatic representation of substrate manufacture. (i) Oligonucleotides were annealed to form double-stranded molecules with A plus 24mer (as shown here) or B plus 24mer. In each case the 24mer was cisplatinated (cis). The GTG cisplatination site is indicated by a triangle and EcoRI compatible overhang shown in boldface. Oligonucleotides A and B were phosphorylated at the 50 end. (ii) These double-stranded molecules were then ligated onto EcoRI linearized pGEM3zf+ plasmid DNA (indicated by horizontal lines and containing no other cisplatin adducts). Acis and Bcis are shown here, Acon and Bcon were constructed identically but lacked the 1,3-d(GpTpG) cisplatin adduct at the GTG cisplatination site. The DNA substrates Acis and Bcis had self-incompatible ends but were compatible with each other. (iii) Position of HindIII site and polarity of exonuclease III digestion. The 12 base fragment resistant to digestion is indicated. (B) Exonuclease III analysis of substrates to confirm cisplatin adduct presence. 32P-end-labelled DNA substrate was subject to restriction enzyme digestion with HindIII, subsequent exonuclease III digestion and denaturing acrylamide (15%) gel electrophoresis. Control Bcon substrate (lanes 1 and 2), cisplatinated Bcis substrate (lanes 3 and 4), oligonucleotide B annealed to control 24mer (lane 5), cisplatinated oligonucleotide B annealed to 24mer (lane 6), 10 bp molecular weight marker (lane 7). Exonuclease III digestion was for 0 h (lanes 1 and 3) and for 2 h (lanes 2 and 4–6). The arrow shows the position of the 12 base fragments (which migrated at 13 bases owing to the presence of the cisplatin adduct) remaining owing to the blockage of nuclease action by the cisplatin adduct. single cisplatin adducts or has used cell extracts that contain all factors required for NHEJ and, more importantly, are at the correct cellular concentration and relative molar ratios to each other; hence, our system is more relevant to the in vivo situation. When treated with a high concentration of cisplatin, large numbers of different adduct types were formed throughout the entire substrate DNA, which led to a dramatic decrease in NHEJ efﬁciency. As the cisplatin concentration was decreased the adduct levels would decrease resulting in the reduction of NHEJ inhibition. However, the actual adduct types and levels formed were difﬁcult to control in the individual plasmid molecules in these reactions. The reduction in end-joining could be due to multiple effects of cisplatin including the physical presence of cisplatin at or near the DNA ends so inhibiting end processing and/or ligation of ends, or to the distortion and denaturation of the DNA helix caused by the cisplatin adducts present along the DNA. Another possibility was that the recruitment of NER proteins to the cisplatin adduct may hinder NHEJ. However, preliminary data using the GM2246 cell line which is deﬁcient in XPC, a protein known to be recruited early in the NER, suggested this last possibility was not likely (data not shown). Therefore, we designed further experiments to help discrim- inate the effects of terminal cisplatin adducts from other events associated with cisplatin treatment. We investigated the use of substrates with different lengths of inserted AT-rich sequences that did not contain the sequence GG or AG such that the most common cisplatin adducts [the intrastrand 1,2-d(GpG) and 1,2-d(ApG) adducts] would not be formed in this region of DNA near the DSB. We reasoned that these substrates could be useful intermediates between using the ‘globally’ cisplat- inated substrate (Figure 1), the effects of which are difﬁcult to interpret, and the single adduct substrates (Figure 4), which are more technically demanding and time-consuming to produce. Although the longer AT-rich substrates were less sensitive to the effect of cisplatin, surprisingly the presence of a single- terminal GTG nucleotide sequence as a target site for cisplat- ination had no signiﬁcant effect on the NHEJ efﬁciency. It is possible that as these substrates contained purines and a GA dinucleotide near the DSB, cisplatin monoadducts and 1,2 intrastrand adducts could be formed; however, it is more likely that the distortion of the DNA structure by cisplatination at multiple sites throughout the rest of the substrate was such that the effect of a single GTG nucleotide sequence as a target site for cisplatination could not be measured. Another possibility is that the GTG sequence may not have been cisplatinated in all substrate molecules owing to the large amount of other poten- tial sites for global cisplatination. In addition, the actual length of DNA over which the DNA-PKcs/Ku heterodimer complex interacts, or that Ku translocates on recruitment of XRCC4/ ligase IV to the DNA ends (19) may be longer than the long AT-rich regions by a further 20 bp, and so would extend into the vector sequence that would contain multiple cisplatin