further as although it makes up only 5–10% of adducts from cisplatin-treated DNA, it is able to both bend and unwind the DNA; therefore, causing substantial distortion of the DNA helix [reviewed in (1)]. Once again, NHEJ by MO59K extract was much reduced with increasing cisplatination concentration (data not shown). Comparison of the effect of cisplatination on the shortcis substrate revealed a decrease in percentage ligation products from 66 – 11% (–SEM) (buffer treated) to 18 – 5% (cisplatinated) at 2 h. However, the same decrease was also observed when the shortcon substrate (in which the terminal GTG site was absent) was treated with cisplatin (70 – 10%, buffer treated and 18 – 4%, cisplatinated) (Figure 2B). The ligation observed with both substrates was DNA-PKcs depend- ent, as judged by Wortmannin sensitivity, and Ku-dependent, using an anti-Ku antibody (data not shown). NHEJ of long AT-rich substrates The DNA substrates shortcon and shortcis had 13 and 15 bases of the AT-rich sequence on either side of the DSB, which is sufﬁcient for a single Ku heterodimer to bind to. But following DNA-PKcs recruitment, the Ku heterodimer is predicted to translocate away from the DSB and along the DNA by a further 10 bases. This would mean that the translocated Ku heterodimer would now also cover the original plasmid DNA sequence which contained many potential cisplatination sites. To minimize the possibility that cisplatin residues other than the one 10 bases from the DSB site were inhibiting Ku trans- location and hence NHEJ, two new substrates were engineered with an extra 16 and 20 bases of AT-rich sequence on either side (sequence in Materials and Methods and Figure 2C). Similar to the effect seen with the short AT-rich substrates, cisplatination of both control (longcon) and cisplatin-site containing (longcis) substrates decreased ligation product formation to the same extent. Percentage ligation for longcon substrate was 69 – 10 (buffer treated) and 36 – 4% (cisplat- inated), while for longcis substrate was 63 – 11 (buffer treated) and 31 – 2% (cisplatinated). However, we did note that both the longer AT-rich substrates (longcon and longcis) were signiﬁcantly less sensitive (P < 0.05) to the effects of cisplat- ination than the short AT-rich substrate; percentage ligation when cisplatinated was 36 – 4% (longcon), 31 – 2% (longcis), 18 – 4% (shortcon) and 18 – 5% (shortcis) at 2 h. NHEJ of single 1,3-d(GpTpG) cisplatin adduct substrates In patients receiving concurrent chemoradiation, the doses of cisplatin and radiation are such that we would not expect clusters of cisplatin adducts in the DNA nor multiple cisplatin adducts near each DSB (3). As it was possible that the addi- tional monoadducts and the GA adduct within the AT-rich region as well as global cisplatination inhibited NHEJ irre- spective of the presence or absence of a single-terminal cisplatin adduct, we investigated the joining of linear DNA substrates containing one cisplatin adduct in the entire molecule. DNA molecules (Acis and Bcis) were manufactured whereby a 1,3-d(GpTpG) cisplatin adduct was located 10 bases from each DNA end. In parallel, control substrates (Acon and Bcon) were made which were not cisplatinated. The 4 nt overhangs of the DNA substrates were self-incompatible, so that in reactions containing each substrate alone end-joining Nucleic Acids Research, 2005, Vol. 33, No. 8 2535 could only occur between incompatible ends. However, substrates A and B contained 4 nt 30 overhangs that were compatible with each other such that, in the presence of both substrates A and B, compatible end-joining could take place (Figure 3A). Analysis using exonuclease III conﬁrmed that the cis substrates contained the cisplatin adduct. Exonuc- lease III acts upon 50 overhangs on double-stranded DNA, but has no activity at 30 overhangs. Following HindIII digestion of the DNA substrates, which creates a 50 overhang, exonuclease III will digest the cisplatin adduct-containing strand starting from the HindIII site and moving towards the engineered DSB. The presence of a cisplatin adduct would impede the activity of exonuclease III and would be predicted to result in a 12 bp fragment (with a 4 base single-strand overhang) resistant to digestion. An example of an exonuclease III digestion of a DNA substrate is shown in Figure 3B. Here, the DNA con- taining the cisplatin adduct is digested from the HindIII- generated 50 overhang towards the 30 end of the molecule, leaving a fragment of 13 base length (which migrated more slowly owing to the cisplatin adduct), whereas the non-cisplatinated control DNA produces smaller fragments (Figure 3B). NHEJ experiments were performed with combinations of these substrates and showed that the DNA containing 1,3-d(GpTpG) cisplatin at both ends was less readily joined than the non-cisplatinated control DNA, with ligation of 26% (Acis plus Bcis) compared with 37% (Acon plus Bcon) of substrate, respectively (Figure 4); a decrease in ligation efﬁ- ciency of 30%. In total, four batches of both cisplatinated and control substrate molecules were independently produced. The actual levels of joining of the control substrates were lower than those observed with the PstI cut substrate in Figure 1 and the short and long AT-rich DNA substrates (35% ligated pro- ducts compared with 70 and 55–60%, respectively). This may reﬂect the multiple processing steps required to make the substrate. In vivo it is more probable that a single cisplatin adduct would occur on one side of a DSB rather than two cisplatin adducts, one on either side of a DSB; experiments were performed to mimic this situation using mixtures of control and cisplatinated substrates with only one end of the DNA containing a cisplatin adduct (i.e. Acon plus Bcis or Bcon plus Acis) (Figure 4). Here, levels of NHEJ were intermediate between the control DNA and the DNA sub- strates cisplatinated at both ends. This joining was DNA- PKcs dependent and Ku dependent (data not shown). We also carried out experiments with the individual substrates alone which have self-incompatible DNA ends. Again, the majority of joining was both DNA-PKcs and Ku dependent (Figure 4C). The NHEJ levels were lower than when comple- mentary ends were present; this was as expected as incom- patible end-joining is known to be a less-efﬁcient process (24). Interestingly, the cisplatinated DNA with incompatible ends was joined with lower efﬁciency than the corresponding control DNA with incompatible ends (Figure 4A). DISCUSSION Using cell-free extracts, we have investigated the effects of cisplatin adducts on the repair of DSBs by the NHEJ pathway. This system is an advance in our understanding of this process as no other study has used long (3.2 kb) DNA substrates with