The complete pattern of mutagenesis arising from the repair of site-specific psoralen crosslinks: analysis by oligonucleotide hybridizatlon

The mutation sites detected by a physical method follow a broader distribution than those identified genetically, and occur primarily at favored psoralen crosslinking sites, where T-T and T-C interstrand crosslinks can be formed.

Abstract

Psoralen crosslinks were site-specifically placed in plasmid pBR322 near the BamHI site in the tet gene by enzymatically inserting mercurated nucleotides and reacting at the target site with a sulfhydryl-containing psoralen. The damaged plasmid was repaired in SOS-induced E. coli cells. Mutants were detected by colony hybridization to oligonucleotides in the target region, and their sequences were determined. The mutations are all base substitutions, 80% transitions and 20% transversions, similar to the mutations previously identified by the loss of tetracycline resistance. However, the mutation sites detected by a physical method, unconstrained by phenotypic changes, follow a broader distribution than those identified genetically. They occur primarily at favored psoralen crosslinking sites, where T-T and T-C interstrand crosslinks can be formed. A majority of these mutations are silent.