Effect of Methyl Methanesulphonate on the Nucleoid Structure of Escherichia Coli

Incubation of a strain of Escherichia coli K12 with 25 mM-methyl methanesulphonate (MMS) for 1 h changed the sedimentation coefficient of the nucleoids from 1600S to 850S. When isolated nucleoids were treated with MMS under identical conditions in vitro there was no change in the sedimentation coefficient. Alkaline sucrose-gradient centrifugation of DNA from cells treated with 25 mM-MMS for 1 h indicated that there were approximately 100 breaks plus apurinic sites per chromosome. Titration with ethidium bromide of nucleoids from MMS-treated cells showed that almost all supercoiling had been lost, suggesting that the breaks plus apurinic sites consisted mostly of breaks. Further experiments showed that the apurinic sites were probably created by non-enzymic depurination and that little non-enzymic strand breakage had occurred. The depurinated sites thus created could then serve as substrates for the apurinic-specific endonucleases of the cell, with the result that strand breakage occurred. MMS treatment did not cause any changes in the DNA:RNA ratio of the nucleoids. Removal of MMS followed by a period of incubation resulted in a decrease in the number of breaks plus apurinic sites and an increase in the sedimentation coefficient of the nucleoids. After 2 h incubation in MMS-free medium the sedimentation coefficient of the nucleoids from MMS-treated cells was the same as that of the control; the supercoiling was also partially restored. The effect of MMS on two MMS-sensitive mutants of E. coli, one a polA and the other a recA mutant, was also studied. In both cases MMS caused complete collapse of the nucleoid structure.