Excision repair at individual bases of the Escherichia coli lacI gene: relation to mutation hot spots and transcription coupling activity.

Abstract

To determine whether base-to-base variations in the rate of excision repair influence the distribution of mutations, we have developed a method to measure UV photoproducts at individual nucleotides in the Escherichia coli chromosome. Specific gene fragments are 3' end-labeled using a sequence-specific oligonucleotide to direct the site of labeling, and photoproducts are identified by enzymatic incision. On the nontranscribed strand of the E. coli lWd gene, the cyclobutane pyrimidine dimer frequency was 2-to 8-fold higher in chromosomal DNA than in a cloned DNA fragment. The chromosomal lesion frequency corresponded to the frequency of UVinduced mutations at mutation hot spots reported in the literature. Only 0-30% of cyclobutane dimers at various sites on this strand were excised in 20 min. In contrast, repair on the transcribed strand was 80-90% complete in 20 min. However, the transcribed strand contained an excision repair "slow spot" at the site of its single mutation hot spot: At this site, no repair occurred for the first 10 min, after which repair proceeded more slowly than typical of that strand. In an mid strain, deficient in a factor that couples repair to transcription in cell extracts, the excision rate at individual nucleotides on the transcribed strand was minimal at most sites for at least 30 min.

DNA damage by anti-cancer agents resolved at the nucleotide level of a single copy gene: evidence for a novel binding site for cisplatin in cells

“…It is however possible that the sequence environment affects the rate of repair of individual lesions. For example, in the bacterial lac I gene it has been demonstrated that, after UV damage, lesions at certain sites are repaired slowly and that these repair 'slow spots' correlate with the positions of mutation 'hot spots' (34). Thus the precise position of genetic mutations caused by carcinogens may be determined by both the distribution of the initial damage and by the efficiency of the subsequent repair of individual lesions.…”

Tumor Suppressor Gene Mutations and Photocarcinogenesis

“…Those that are also hotspots in internal cancers lie at important structural sites of the p53 protein (21). Those that are hotspots only in skin cancers may reflect slow repair of UV photoproducts at specific base pairs (22,23). The two internal-cancer hotspots that are not skin cancer hotspots are located at pyrimidines flanked by purines; UV photoproducts should not form at these sites and mutations should not be found in skin tumors.…”

Transcription-repair coupling and mutation frequency decline

“…Most mutations in an active gene arise from lesions in the nontranscribed strand in wildtype cells and from lesions in the transcribed strand in mfd mutant cells (29), presumably because lesions in the template strand are repaired relatively rapidly in wild-type cells and lesions in the coding strand are repaired more efficiently in mfd mutant cells (20).…”