Figure 4. NHEJ efficiency in control and 1,3-d(GpTpG) cisplatinated DNA. (A) Representative NHEJ agarose gel of DNA substrates either uncisplatinated (Acon + Bcon), 1,3-d(GpTpG)-cisplatinated at both ends (Acis + Bcis), 1,3- d(GpTpG)-cisplatinated at one end (Acon + Bcis and Acis + Bcon) or single substrates alone. Reactions were incubated for 2 h at 37 C. The 3.2 kb linear monomer (1·) was joined to form ligated linear dimers (2·) and higher multimers (m) as indicated. MW indicates 1 kb molecular weight markers. (B) Corresponding graph showing the percentage ligation formation. The aver- age of three independent experiments (–SEM) is shown. (C) End-joining of self-incompatible DNA substrates in the presence (+) or absence ( ) of Wortmannin (10 mM) or anti-Ku70 antibody (1:20 dilution). Linear monomer (1·) and ligated linear dimers (2·) are indicated. adducts. Therefore, multiple internal cisplatin adducts further away from the termini, which could cause DNA distortion, have a more profound effect on NHEJ such that we were not able to measure the effect of terminal cisplatin adducts on NHEJ using these intermediate substrates. Indeed, the longer AT-rich constructs were less sensitive to the effects of global cisplatination than the shorter constructs as the extra 16–20 bp Nucleic Acids Research, 2005, Vol. 33, No. 8 2537 AT sequence inserted into the longer constructs, where there was vector sequence before in the shorter constructs, again reinforced the effect of multiple cisplatin adduct sites further away from the actual DNA termini on NHEJ. We then analysed the effect on NHEJ of a single adduct at both ends of the DSB in a molecule with no other adducts present, and the effect when only one DSB end had a cisplatin adduct. The latter situation is more likely to occur in vivo. NHEJ efﬁciency was reduced when both ends contained cisplatin adducts by 30%, while a reduction of 20% was found when a cisplatin adduct was present near only one DNA end. It is not possible to directly compare the percentage end-joining of substrates between experiments as substrates with different terminal polarity and sequence were used. Additionally, in experiments with substrates that had single cisplatin adducts there was a mixture of compatible and incompatible DNA ends, which were not phosphorylated. End-joining efﬁciency can alter depending on the polarity of DNA ends, the nucleotide sequence at the termini or the nucleotide sequence internal to the DNA ends (21,25–27); we have shown this in our work where termini differing by only two bases were joined with different efﬁciencies (Acis com- pared with Bcis; Figure 4B). This can be due to the differential activation of DNA-PK. However, the results with global plat- ination of substrate showed that, using the highest dose of cisplatin, total NHEJ inhibition could be achieved (Figure 1) where the adducts would be both internal and at the DNA ends. The multiple cisplatin adducts situated more internally in the AT-rich substrates (Figure 2) also were able to substantially reduce NHEJ at the cisplatin concentrations used, and at higher levels of cisplatin could block NHEJ even more effect- ively (data not shown). This indicated that multiple internal adducts could have more inﬂuence on NHEJ than a single terminal adduct, which caused a maximal 20–30% inhibition (Figure 4). Similarly, it is likely that the multiple internal adducts in the substrate used in Figure 1 could have more inﬂuence on NHEJ than the single GG adduct site at the extreme DNA end. This reinforces the importance of our study using single deﬁned adducts and the difﬁculties inherent in the interpretation of studies that use globally cisplatinated substrates. These data argue against using globally cisplatin- ated DNA substrates for determining the effect of terminal cisplatin adducts on the NHEJ. While this work was under review, studies by Pawelczak et al. (26) were published that conﬁrmed the inhibitory effect on part of the NHEJ pathway of cisplatin adducts close to the DSB. Using puriﬁed DNA-PKcs and recombinant Ku they showed that DNA-PK kinase activity, but not Ku binding, was signiﬁcantly inhibited (95%) by the presence of a 1,2-d(GpG) intrastrand cisplatin adduct 6 bp from the DNA terminus, while an adduct 15 bp away caused partial inhibition (60%). In view of the different termini used and adduct types, it is not possible to directly compare levels of inhibition with this work. The authors propose a model whereby a cisplatin adduct close to the terminus prevents the DNA substrate from fully thread- ing through the kinase such that the single-stranded ends would not be positioned in the activating cavity of DNA- PKcs so preventing end-joining. Our ﬁndings agree with others in the ﬁeld who suggest that cisplatin adducts can decrease NHEJ due to a decrease in the translocation rate of Ku and the consequent decrease in DNA-PK kinase activity (16–19).