Uracil incorporation into nascent DNA of thymine-requiring mutant of Bacillus subtilis 168.

Abstract

A thymine-requiring mutant of Bacillus subtilis strain 168 accumulates short DNA chains after brief pulses with [(3)H]thymidine. Reversion of the thy mutation to thy(+) abolishes the accumulation of short DNA chains, suggesting that the accumulation is related to the thy mutation. The reason for this accumulation has been further investigated by analysis of a mutant with a defective uracil-DNA glycosidase activity (urg). The accumulation of short DNA chains in thy(-) cells is abolished by the deficiency of uracil-DNA glycosidase activity. In thy(+) cells, the deficiency of the glycosidase activity does not change the sedimentation profile of pulse-labeled DNA. DNA isolated from thy(-)urg(-) cells is fragmented by successive treatment with purified uracil-DNA glycosidase and alkali, indicating that uracil residues are present in this DNA. DNA isolated from thy(+)urg(-) cells is not fragmented by the same treatment. Significant radioactivity is detected in the dUMP region, when [(3)H]uridine-labeled DNA from thy(-)urg(-) cells is hydrolyzed and analyzed by thin-layer chromatography. Only a trace amount of radioactivity, which is not influenced by the deficiency of uracil-DNA glycosidase activity, is found in the dUMP region in DNA hydrolysates from thy(+) cells. These results suggest that, in thy(-) cells, uracil is incorporated into DNA and the accumulation of short DNA chains results from the excision-repair of this uracil whereas in thy(+) cells, uracil is seldom, if ever, incorporated into DNA.