2538 Nucleic Acids Research, 2005, Vol. 33, No. 8 However, assay systems distinct to ours were used by these authors including shorter lengths of substrate DNA, sometimes as low as 32 nt, and higher levels of Ku molecules. The rel- evance of having multiple Ku complexes translocating along the DNA strand in vivo is not known, as in vitro a single Ku heterodimer is sufﬁcient to recruit DNA-PKcs (11), which could indicate that in vivo a functional stoichometry is 1:1 for each DNA end. It is also unclear if DNA-PKcs is present in one assay system (19). Our assay system not only provides a measure of the entire Ku and DNA-PK dependent NHEJ pro- cess in a more physiological setting but also allowed us to study the effect of a single adduct, which is more relevant to the situation in vivo (3). In mammalian cells, ionizing radiation causes 40 DSBs per cell per gray of radiation and, following cisplatin treat- ment, one would expect 1–2 · 104 cisplatin adducts per cell given a total of 3.3 · 109 bp per cell [based on the data and the calculations from (3,28)]. In chemoradiation treatment, the patient is pretreated with cisplatin before the radiotherapy is administered so the assumption is made that cisplatin adducts are present before the DSB are created. Therefore after 2 Gy of radiation, Poisson statistics (3) predict there to be a 0.5–1% probability of a cell having a single cisplatin adduct within 10 bp of one end of a DSB, while the probability of a cell having a cisplatin adduct at 10 bp from both ends of the DNA involved in a DSB is inﬁnitesimally small. We have shown that the presence of a cisplatin adduct at one end reduces the NHEJ efﬁciency at 2 h by 20%. This equates to approximately one in ﬁve end-joining events being delayed/ inhibited by the presence of the cisplatin adduct. Therefore in a cell population treated with chemoradiation as described, we would expect 0.1–0.2% (one-ﬁfth of 0.5–1%) to have DSB rejoining delayed/inhibited by the presence of a cisplatin adduct within 10 bp of one DSB end, a potentially deleterious event as only one unrepaired DSB needs to remain within the cell to initiate cell death. Repair of DSB induced by ionizing radiation is efﬁcient, such that in vitro all but 10% of DSB are repaired within 2 h of radiation (29), so any reduction in efﬁciency of repair by the presence of a cisplatin adduct could impact on treatment outcome. The relative importance of NHEJ and HR in the repair of DSB with a cisplatin adduct close by in vivo is not known. In terms of cisplatin treatment alone, cisplatin exposure has no effect on NHEJ-deﬁcient cells, whereas HR-deﬁcient cells are more sensitive (30,31). This is due to the decrease/stalling of replication fork progression by the presence of the cisplatin adduct, which may lead to the production of DSBs [(32,33) discussed in (34)]. This mechanism may be especially import- ant in relation to the type of adduct formed as the most fre- quent adduct, GpG, is refractory to repair by NER, while the less-frequent GpNpG adduct is more efﬁciently repaired by NER (35). In addition, interstrand cross-links formed by cis- platin adducts require HR for their repair (36). Therefore, although we have shown that NHEJ is impeded by the pres- ence of a nearby cisplatin adduct, thus increasing radiosensit- ivity, it is likely that a proportion of the radiosensitizing effect of cisplatin is related to the HR pathway. We have shown that a single cisplatin adduct, either at one or both ends of the DNA substrate, can decrease the joining of compatible DNA ends. The MO59K cell line has previously been shown to join incompatible DNA ends by NHEJ (24) and in this study we have shown that incompatible ends are joined less efﬁciently if they contain a single cisplatin adduct. Our experiments with exonuclease III indicate that exonuclease movement along the DNA strand can be blocked by the presence of a single cis- platin adduct, and it is known that the presence of adducts can block DNA polymerases a, e and d (37,38), RNA polymerase II and T7 RNA polymerase (39), although DNA polymerase b can bypass adducts (38). This hinderance of nuclease and polymerase action is likely to play a signiﬁcant role in the inhibition of repair processes following concurrent chemora- diation, where the ends of DSBs produced in response to irradiation are unlikely to be compatible and may need to be trimmed and ﬁlled-in prior to ligation. In conclusion, using a cell-free extract system and an assay for NHEJ we have shown that the presence of a single cisplatin adduct near a DSB reduces NHEJ efﬁciency and that this occurs at cellular cisplatin adduct levels commensurate with clinical doses of concurrent chemoradiation. Therefore, one mechanism by which cisplatin may exert its radiosensit- izing effect is by reducing the efﬁciency of NHEJ. In addition, this work demonstrates the limitations of the use of DNA substrates containing any cisplatin adducts internal to the DNA ends for determining the effect of cisplatinated substrate termini on NHEJ. ACKNOWLEDGEMENTS Many thanks to Dr Tomas Lindahl for provision of laboratory space and Colin Johnston for help with the statistical analysis. Financial support was provided by Cancer Research UK. Funding to pay the Open Access publication charges for this article was provided by JISC and Cancer Research UK. Conflict of interest statement. None declared. REFERENCES 1. 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