Nucleic Acids Research, 2005, Vol. 33, No. 8 2531–2539 doi:10.1093/nar/gki528 Inhibition of double-strand break non-homologous end-joining by cisplatin adducts in human cell extracts C. P. Diggle, J. Bentley, M. A. Knowles and A. E. Kiltie* Cancer Research UK Clinical Centre, St James’s University Hospital, Leeds LS9 7TF, UK Received December 2, 2004; Revised February 25, 2005; Accepted April 4, 2005 ABSTRACT The effect of cis-diaminedichloroplatinum(II) (cis- platin) DNA damage on the repair of double-strand breaks by non-homologous end-joining (NHEJ) was determined using cell-free extracts. NHEJ was dra- matically decreased when plasmid DNA was damaged to contain multiple types of DNA adducts, along the molecule and at the termini, by incubation of DNA with cisplatin; this was a cisplatin concentration- dependent effect. We investigated the effect a single GTG cisplatination site starting 10 bp from the DNA termini would have when surrounded by the regions of AT-rich DNA which were devoid of the major adduct target sequences. Cisplatination of a substrate con- taining short terminal 13–15 bp AT-rich sequences reduced NHEJ to a greater extent than that of a sub- strate with longer (31–33 bp) AT-rich sequences. However, cisplatination at the single GTG site within the AT sequence had no significant effect on NHEJ, owing to the influence of additional minor monoad- duct and dinucleotide adduct sites within the AT-rich region and owing to the influence of cisplatination at sites upstream of the AT-rich regions. We then studied the effect on NHEJ of one cis-[Pt(NH3)2{d(GpTpG)- N7(1),-N7(3)} [abbreviated as 1,3-d(GpTpG)] cisplatin adduct in the entire DNA molecule, which is more reflective of the situation in vivo during concurr- ent chemoradiation. The presence of a single 1,3- d(GpTpG) cisplatin adduct 10 bases from each of the two DNA ends to be joined resulted in a small (30%) but significant decrease in NHEJ efficiency. This process, which was DNA-dependent protein kinase and Ku dependent, may in part explain the radiosensitizing effect of cisplatin administered during concurrent chemoradiation. INTRODUCTION Concurrent chemoradiation, the use of chemotherapy during a course of radiotherapy, is now the standard treatment in several cancer types and the subject of clinical trials in others. However, the molecular mechanisms that make this an effective treatment are not fully understood. cis-Diaminedi- chloroplatinum(II) (cisplatin), an agent commonly used in chemoradiation, acts by producing DNA interstrand and intrastrand adducts. In vitro, 65% of the adducts are cis- [Pt(NH3)2fd(GpG)-N7(1),-N7(2)g] [abbreviated as 1,2-d(GpG)] intrastrand cross-links, 25% cis-[Pt(NH3)2fd(ApG)-N7(1), -N7(2)g] [abbreviated as 1,2-d(ApG)] intrastrand cross-links, 5–10% 1,3-d(GpNpG) intrastrand cross-links, with a small per- centage of interstrand cross-links and monofunctional adducts [reviewed in (1)]. The intrastrand adducts are removed by nuc- leotide excision repair (NER), which involves excision of a region of 30 nt around the adduct, followed by DNA synthesis and ligation (2). Radiation exposure can produce many types of DNA damage including base damage, single-strand breaks and double-strand breaks (DSBs). In a locally multiply damaged site where a cisplatin adduct occurs close to a single-strand break the damage may be initially repaired by NER. However, during the dual incision step a DSB, which is the most lethal type of damage a cell can sustain, could be produced (3). Alter- natively, if a cisplatin adduct is close to a DSB, the DSB could physically block the cisplatin removal and repair by the NER pathway, or the cisplatin adduct could hinder the DSB repair pathways by restricting the access of the protein complexes involved. Another possibility is that the NER and the DSB repair proteins may compete for access to the locally multiply damaged site. Investigations into the efﬁciency of NER have shown that NER is less efﬁcient if damaged sites [cisplatin adducts and ultraviolet (UV) lesions] are near to a DSB owing to binding of the DSB repair protein heterodimer Ku70/80 at the DNA ends (4). In addition, decreased levels of NER proteins bind to the lesion if a DSB is present (5,6). The DSBs are repaired by the homologous recombination (HR) pathway or the non-homologous end-joining (NHEJ) *To whom correspondence should be addressed. Tel: +44 113 206 4908; Fax: +44 113 242 9886; Email: anne.kiltie@cancer.org.uk Correspondence may also be addressed to C. P. Diggle. Tel: +44 113 206 5674; Fax: +44 113 244 4475; Email: c.p.diggle@leeds.ac.uk ª The Author 2005. Published by Oxford University Press. All rights reserved. The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions@oupjournals.org