2534 Nucleic Acids Research, 2005, Vol. 33, No. 8 concentration used. Examination of the DNA sequence at the PstI DNA ends showed a large number of potential sites along the molecule where the cisplatin could form an adduct (sequence in Materials and Methods). For example, both ends of this substrate contained a GpG site, which if cisplatinated could lead to fraying of the DNA ends (23), AG sites were identiﬁed at 11 and 15 bp from the DNA ends, and potential interstrand cross-links at 2/3, 6/7 and 10/11 bp on one side and 4/5 bp on the other side. Therefore, to distinguish the effect of terminal cisplatin adducts from other cisplatin-mediated effects on end-joining we constructed substrates with con- trolled sites for cisplatination at the DNA ends. In a prelimin- ary experiment we generated plasmid DNA cut with a second restriction enzyme, Aﬂ III, which did not have any G bases at the DNA ends, but did contain one GTG sequence adjacent to the 30 cohesive overhang. Once again a decrease in NHEJ was seen with increasing cisplatination (data not shown). NHEJ of short AT-rich substrates We reasoned that the introduction of one deﬁned GTG cisplat- ination site at the termini of the substrate, which would be surrounded by DNA containing a reduced number of potential cisplatination sites, would allow us to determine the effect of a terminal cisplatin adduct on NHEJ. We engineered two new substrates (shortcon and shortcis) with fewer potential cisplat- ination sites as no GG, AG or GNG sequences were present in the immediate sequence surrounding the DSB owing to the insertion of a 13 and 15 bp AT-rich DNA sequence on either side of the DSB (sequence in Materials and Methods and Figure 2A). Both substrates were linearized with MfeI and the only difference between them was that the ‘shortcis’ sub- strate had a GTG sequence inserted 10–12 bases from the restriction site DSB restriction site. This GTG sequence allowed the production of a 1,3-d(GpTpG) intrastrand cisplatin adduct. This adduct was speciﬁcally selected to investigate Figure 2. NHEJ of short and long AT-rich substrates. Sequence of the ends of the (A) short and (C) long AT-rich control and cisplatinated DNA substrates. Double- stranded oligonucleotides were ligated into pGEM plasmid DNA (underlined) with the MfeI restriction site shown in boldface. The inserted triangles show the position of the GTG sequence for cisplatination. Graphs (B) and (D) show the percentage of ligation products formed by MO59K extract and DNA substrates shown in (A) and (C), respectively. Substrates were either buffer treated or cisplatinated (2.65 nmol cisplatin). Experiments were performed in triplicate (–SD) and are representative of three independent experiments.