Hierarchy of lesion processing governs the repair, double-strand break formation and mutability of three-lesion clustered DNA damage

Abstract

Ionising radiation induces clustered DNA damage sites which pose a severe challenge to the cell's repair machinery, particularly base excision repair. To date, most studies have focussed on two-lesion clusters. We have designed synthetic oligonucleotides to give a variety of three-lesion clusters containing abasic sites and 8-oxo-7, 8-dihydroguanine to investigate if the hierarchy of lesion processing dictates whether the cluster is cytotoxic or mutagenic. Clusters containing two tandem 8-oxoG lesions opposing an AP site showed retardation of repair of the AP site with nuclear extract and an elevated mutation frequency after transformation into wild-type or mutY Escherichia coli. Clusters containing bistranded AP sites with a vicinal 8-oxoG form DSBs with nuclear extract, as confirmed in vivo by transformation into wild-type E. coli. Using ung1 E. coli, we propose that DSBs arise via lesion processing rather than stalled replication in cycling cells. This study provides evidence that it is not only the prompt formation of DSBs that has implications on cell survival but also the conversion of non-DSB clusters into DSBs during processing and attempted repair. The inaccurate repair of such clusters has biological significance due to the ultimate risk of tumourigenesis or as potential cytotoxic lesions in tumour cells.

Figure 1.

Repair of an AP site when present in a cluster with two tandem 8-oxoG lesions on the opposing strand. (A) Repair of the AP site after incubation with CHO-K1 nuclear extract. (B) Accumulation of DNA in the intermediate repair bands (SSB+1 and SSB+2): (circle) AP control, (square) AP/8-oxoG+1+5, (diamond) AP/8-oxoG+2−2, (triangle) AP/8-oxoG−1−5. Error bars represent the standard error of the mean from at least three independent experiments.

Figure 2.

Prompt formation of DSBs where incision in clusters containing bistranded AP sites, of either of the bistranded AP sites is not inhibited. (A) Repair of the AP site after incubation with CHO-K1 nuclear extract. (B) Formation of DSBs: (circle) AP control, (square) AP/AP+5 8-oxoG+1, (diamond) AP/AP−5 8-oxoG−1, (triangle) AP/AP−2 8-oxoG+2, (cross) AP/AP−1 8-oxoG−5. Error bars represent the standard error of the mean from at least three independent experiments.

Figure 3.

Slow DSB formation in clusters containing bistranded AP sites, when the incision of the AP site in tandem with 8-oxoG confers retardation of incision on the opposing, single AP site. (A) Repair of the AP site after incubation with CHO-K1 nuclear extract: (filled circle) AP control, (filled square) *AP/AP+1 8-oxoG+5, (filled triangle) *AP/AP+2 8-oxoG−2, (open square) AP/*AP+1 8-oxoG+5, (open triangle) AP/*AP+2 8-oxoG−2. The ³²P-labelled strand is denoted by asterisk in each case. (B) Formation of DSBs: (circle) AP control, (square) AP/AP+1 8-oxoG+5, (triangle) AP/AP+2 8-oxoG−2. Error bars represent the standard error of the mean from at least three independent experiments.

Figure 4.

E. coli colony survival after transformation with clustered damage sites. (A) Colony survival after transformation of clustered DNA damage into wild-type E. coli. Mean colony numbers: no lesion control (120 650), U control (100 050), 8-oxoG control (115 650), U/U+5 8-oxoG+1 (4000), U/U−1 8-oxoG−5 (0), U/U+1 8-oxoG+5 (31 125), U/U+2 8-oxoG−2 (2000), U/8-oxoG+1 +5 (173 000), U/8-oxoG+2 −2 (157 000), U/8-oxoG−1 −5 (164 312.5). (B) Colony survival after transformation of clustered DNA damage into ung1 E. coli. Mean colony numbers: no lesion control (65 000), U control (46 333.3), AP control (38 000), U/U+5 8-oxoG+1 (66 250), AP/AP+5 8-oxoG+1 (4000), U/U-1 8-oxoG−5 (40666.7), U/U+1 8-oxoG+5 (56 333.3), U/U+2 8-oxoG−2 (27 250), AP/AP+2 8-oxoG−2 (3333.3). Clustered damage sites transformed are shown along the horizontal axes. No lesion control refers to an oligonucleotide containing no damage inserted into the plasmid before transformation. Error bars represent the standard error of the mean from at least three independent experiments.