The ATM+/+/p53?/? and ATM?/?/p53?/?, selleck chem inhibitor and the DNA-PK+/+/p53?/? and DNA-PK?/?/p53?/? mouse embryonic fibroblasts, were generously provided by Dr P Leder (Westphal et al, 1997) and Dr EH Goodwin (Bailey et al, 1999), respectively. The cells were maintained in DMEM medium supplemented with 2mM L-glutamine (Life Technologies), 10% heat-inactivated foetal calf serum (Oxoid) and penicillin/streptomycin (100Uml?1/100��gml?1, Life Technologies). All cell lines were tested negative for contamination with Mycoplasma spp. and maintained in a controlled environment of 5% CO2 and 95% relative humidity at 37��C. Except for the ATM+/+/p53?/? and ATM?/?/p53?/? mouse cells, which grow as a monolayer and do not form colonies, all other cell lines used in these experiments form well-defined individual colonies when seeded sparsely on standard tissue culture plates.

Reagents Distamycin A and its derivatives brostallicin (PNU-166196) and tallimustine (PNU-152241) were synthesised by Pharmacia Italy (Nerviano, Italy). The chemical structures of the derivatives are presented in Figure 1. Brostallicin was dissolved in methanol, tallimustine in DMSO, and distamycin A in water. Stock solutions were stored at ?20��C. The final concentration of DMSO or methanol in the cultures was <0.1% at all drug concentrations and in controls. Previous experiments (data not shown) have shown that neither 0.1% DMSO nor 0.1% methanol affects the viability or growth of these cell lines. Figure 1 Chemical structures of brostallicin and tallimustine. Both molecules share the distamycin A backbone.

MPE footprinting analysis The MPE footprinting method has been previously described in detail (Hertzberg and Dervan, 1984). The 4492- and 751-bp fragments of SV40-labelled plasmid previously described (Marchini et al, 1999) were incubated with distamycin A, tallimustine, and brostalicin (50��M) for 1h at room temperature and treated for 30min at room temperature with a solution of MPE-(NH4)2-Fe(SO4)2 (synthesised by Pharmacia, Italy, according to the published method; Hertzberg and Dervan, 1984). After precipitation, DNA was resuspended in loading buffer and electrophoresed on an 8% polyacrylamide 7M urea gel and autoradiographed. Taq polymerase stop assay Studies with the Taq stop assay were based on a previously reported method (Ponti et al, 1991).

Prior to drug-DNA incubation, plasmid pBSSK-TOPO II was linearised with a PstI restriction enzyme (NEB) to provide a stop for the Taq polymerase, downstream from the primer. After drug treatment, the DNA was precipitated and washed as described (Ponti et al, 1991). The primer was 5��-end labelled with T4 polynucleotide kinase (NEB) and [��-32P] ATP (5000Cimmol?1, AV-951 Amersham). The synthetic primer sequence and the linear PCR amplification conditions were performed as described (Marchini et al, 1998).