A system to determine basepair substitutions at the molecular level, based on restriction enzyme analysis; influence of the muc genes of pKM101 on the specificity of mutation induction in E. coli

Abstract

A system has been developed for the analysis of basepair substitutions that are involved in the reversion of a specific missense mutation. The method is based on the ability of restriction enzymes to recognize and cut specific DNA sequences. Wild-type revertants arising from AT → GC transitions, pseudo wild-type revertants arising from AT-transversions and second site revertants can be distinguished. 4 mutagenic agents have been used 2,6-diaminopurine, MMS, EMS and ENU, which differ in the types of damage they cause in DNA and in the susceptibility of the damage to repair. All 4 mutagens effectively enhanced the reversion of the mutation studied, trpA223, particularly by increasing the fraction of AT → GC transitions. In this system the influence of the muc genes of plasmid pKM101 was investigated. The presence of these genes reduced the fraction of AT → GC transitions and enhanced the fraction of AT-transversions as well as the fraction of second-site mutations. This change in mutation specificity is found irrespective whether mutation induction occurs mainly via SOS repair (MMS, ENU) or via mainly misreplication (2,6-diAP, EMS). These data suggest that the muc genes are involved in the induction of mutations not only during SOS repair, but also during misreplication. The change in mutation specificity may be caused by a change in the selection and insertion of nucleotides by the DNA-polymerising complex, or by interference with the repair of mismatched bases.