In the yeast Kluyveromyces lactis, the telomeres are composed of perfect 25-bp repeats copied from a 30-nucleotide RNA template defined by 5-nucleotide terminal repeats. A genetic dissection of the K. lactis telomere was performed by using mutant telomerase RNA (TER1) alleles to incorporate mutated telomeric repeats. This analysis has shown that each telomeric repeat contains several functional regions, some of which may physically overlap. Mutations in the terminal repeats of the template RNA typically lead to telomere shortening, as do mutations in the right side of the Rap1p binding site. Mutations in the left half of the Rap1p binding site, however, lead to the immediate formation of long telomeres. When mutated, the region immediately 3 of the Rap1p binding site on the TG-rich strand of the telomere leads to telomeres that are initially short but eventually undergo extreme telomere elongation. Mutations between this region and the 3 terminal repeat cause elevated recombination despite the presence of telomeres of nearly wild-type length. Mutants with highly elongated telomeres were further characterized and exhibit signs of telomere capping defects, including elevated levels of subtelomeric recombination and the formation of extrachromosomal and single-stranded telomeric DNA. Lengthening caused by some Rap1 binding site mutations can be suppressed by high-copynumber RAP1. Mutated telomeric repeats from a delayed elongation mutant are shown to be defective at regulating telomere length in cells with wild-type telomerase, indicating that the telomeric repeats are defective at telomere length regulation.

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We have examined the mutational specificity of 1-nitroso-6-nitropyrene (1,6-NONP), an activated metabolite of the carcinogen 1,6-dinitropyrene, in the lacI gene of Escherichia coli strains which are deficient in nucleotide excision repair (strain NR6113, delta uvrB; strain CM6114, delta uvrB, plasmid pKM101). Separate collections of lacI- mutations and dominant lacI-d mutations, which contain DNA sequence alterations in the region of the lacI gene that encodes the DNA binding domain of the lacI repressor, were made following 1,6-NONP treatment. The DNA sequence of 418 mutations was determined, of which 228 were lacI- mutations and 190 were lacI-d mutations. Ninety three percent of the induced point mutations occurred at G:C residues.0 -(G:C) frameshifts were the dominant mutational class in the lacI- collections of both NR6113 and CM6114, and in the lacI-d collection of NR6113. The frameshift mutations occurred preferentially in runs of guanine residues and their frequency increased markedly with the length of the reiterated sequence. In strain CM6114, which contained the plasmid pKM101, there was a marked stimulation in the frequency of G:C-->T:A transversions that was particularly apparent in the lacI-d collection. We discuss models which might account for the apparent differences in mutational specificity resulting from the presence of the UmuD/C and MucA/B proteins. The results suggest that major classes of mutation are recovered in both the lacI- and lacI-d collections. However, the proportions of the major classes of mutations within the two collections can differ significantly. Depending on the genetic background of the host strain, the relative ratios of base substitutions to frameshift mutations in the lacI-d target can differ by almost an order of magnitude as compared with the lacI- target. This is primarily a function of the relative mutational target size of the different classes of mutation.

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