ABSTRACT
A multiply damaged site (MDS) is defined as > or =2 lesions within a distance of 10-15 base pairs (bp). MDS generated by ionizing radiation contain oxidative base damage, and in vitro studies have indicated that if the base damage is <3bp apart, repair of one lesion is inhibited until repair of the lesion in the opposite strand is completed. Inhibition of repair could result in an increase in the mutation frequency of the base damage. We have designed an assay to determine whether a closely opposed lesion causes an increase in adenine insertion opposite an 8-oxodG in bacteria. We have positioned the MDS (an 8-oxodG in the transcribed strand and a second 8-oxodG immediately 5' to this lesion in the non-transcribed strand) within the firefly luciferase coding region. During two rounds of replication, insertion of adenine opposite the 8-oxodG in the transcribed (T) or non-transcribed (NT) strand results in a translation termination codon at position 444 or 445, respectively. The truncated luciferase protein is inactive. We have generated double-stranded oligonucleotides that contain no damage, each single 8-oxodG or the MDS. Each double-stranded molecule was ligated into the reporter vector and the ligation products transformed into wild-type or Mut Y-deficient bacteria. The plasmid DNA was isolated and sequenced from colonies that did not express luciferase activity. In wild-type bacteria, we detected a translation stop at a frequency of 0.15% (codon 444) and 0.09% (codon 445) with a single 8-oxodG in the T or NT strand, respectively. This was enhanced approximately 3-fold when single lesions were replicated in Mut Y-deficient bacteria. Positioning an 8-oxodG in the T strand within the MDS enhanced the mutation frequency by approximately 2-fold in wild-type bacteria and 8-fold in Mut Y-deficient bacteria, while the mutation frequency of the 8-oxodG in the NT strand increased by 6-fold in Mut Y-deficient bacteria. This enhancement of mutation frequency supports the in vitro MDS studies, which demonstrated the inability of base excision repair to completely repair closely opposed lesions.
