Inhibition of double-strand break non-homologous end-joining by cisplatin adducts in human cell extracts - PubMed (original) (raw)
Inhibition of double-strand break non-homologous end-joining by cisplatin adducts in human cell extracts
C P Diggle et al. Nucleic Acids Res. 2005.
Abstract
The effect of cis-diaminedichloroplatinum(II) (cisplatin) DNA damage on the repair of double-strand breaks by non-homologous end-joining (NHEJ) was determined using cell-free extracts. NHEJ was dramatically 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 containing short terminal 13-15 bp AT-rich sequences reduced NHEJ to a greater extent than that of a substrate 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 monoadduct 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 concurrent 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.
Figures
Figure 1
NHEJ of control and cisplatinated DNA substrate. End-joining performed with MO59K extract and DNA substrate treated with cisplatin at the following concentration (nmol) (A) 5.3, (B) 4.4, (C) 3.5, (D) 2.65, (E) 1.1 and (F) buffer only. NHEJ was measured over a time course where DNA was incubated with extract for 0, 0.5, 1, 2, 3 and 4 h (lanes 1–6). The 3.2 kb linear monomer (1×) was joined to form ligated linear dimers (2×), trimers (3×) and higher multimers (m) as indicated. (G) Proportion of DNA substrate ligated against time at the indicated cisplatin concentrations.
Figure 2
NHEJ of short and long AT-rich substrates. Sequence of the ends of the (A) short and (C) long AT-rich control and cisplatinated DNA substrates. Double-stranded oligonucleotides were ligated into pGEM plasmid DNA (underlined) with the MfeI restriction site shown in boldface. The inserted triangles show the position of the GTG sequence for cisplatination. Graphs (B) and (D) show the percentage of ligation products formed by MO59K extract and DNA substrates shown in (A) and (C), respectively. Substrates were either buffer treated or cisplatinated (2.65 nmol cisplatin). Experiments were performed in triplicate (±SD) and are representative of three independent experiments.
Figure 3
Manufacture of substrate with a single 1,3-d(GpTpG) cisplatin adduct. (A) Diagrammatic representation of substrate manufacture. (i) Oligonucleotides were annealed to form double-stranded molecules with A plus 24mer (as shown here) or B plus 24mer. In each case the 24mer was cisplatinated (cis). The GTG cisplatination site is indicated by a triangle and EcoRI compatible overhang shown in boldface. Oligonucleotides A and B were phosphorylated at the 5′ end. (ii) These double-stranded molecules were then ligated onto EcoRI linearized pGEM3zf+ plasmid DNA (indicated by horizontal lines and containing no other cisplatin adducts). Acis and Bcis are shown here, Acon and Bcon were constructed identically but lacked the 1,3-d(GpTpG) cisplatin adduct at the GTG cisplatination site. The DNA substrates Acis and Bcis had self-incompatible ends but were compatible with each other. (iii) Position of HindIII site and polarity of exonuclease III digestion. The 12 base fragment resistant to digestion is indicated. (B) Exonuclease III analysis of substrates to confirm cisplatin adduct presence. 32P-end-labelled DNA substrate was subject to restriction enzyme digestion with HindIII, subsequent exonuclease III digestion and denaturing acrylamide (15%) gel electrophoresis. Control Bcon substrate (lanes 1 and 2), cisplatinated Bcis substrate (lanes 3 and 4), oligonucleotide B annealed to control 24mer (lane 5), cisplatinated oligonucleotide B annealed to 24mer (lane 6), 10 bp molecular weight marker (lane 7). Exonuclease III digestion was for 0 h (lanes 1 and 3) and for 2 h (lanes 2 and 4–6). The arrow shows the position of the 12 base fragments (which migrated at ∼13 bases owing to the presence of the cisplatin adduct) remaining owing to the blockage of nuclease action by the cisplatin adduct.
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 average of three independent experiments (±SEM) is shown. (C) End-joining of self-incompatible DNA substrates in the presence (+) or absence (−) of Wortmannin (10 μM) or anti-Ku70 antibody (1:20 dilution). Linear monomer (1×) and ligated linear dimers (2×) are indicated.
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