The effect of trimethylpsoralen — Crosslinks on entry of donor DNA in transformation and transfection of Bacillus subtilis

Abstract

Transforming chromosomal DNA, irradiated with long-wave UV light in the presence of 4,5',8-trimethylpsoralen (TMP) binds to competent B. subtilis cells as effectively as non-treated DNA, but its transforming activity is strongly reduced. Uptake studies show that the entry of transforming DNA, after some stimulation by short periods of irradiation in the presence of TMP, decreases proportionally with the dose of irradiation. Crosslinking was quantitated by electron microscopy. Since the number of crosslinks increases proportionally with the dose of irradiation, it is suggested that entry of donor DNA is prevented by crosslinks. The inhibition of entry of DNA is paralleled both by decreased breakdown of crosslinked DNA interacting with competent cells, and decreased breakdown by nuclease activity liberated during protoplasting of competent cultures. These data support the model of Lacks et al. (1976) which postulates that a membrane-bound deoxyribonuclease is engaged in the entry of donor DNA into the competent cell. The transforming activity of the chloramphenicol-resistance carrying plasmid pC194, originally obtained from Staphylococcus aureus, is also destroyed by TMP crosslinks. Contrary to chromosomal DNA, its association with the cells is stimulated by long-wave UV irradiation in the presence of TMP, but experiments are presented suggesting that the DNA is still vulnerable to the action of exogenous pancreatic deoxyribonuclease. Transfecting SPP1 DNA is also inactivated by TMP crosslinks. Marker rescue of transfecting DNA containing crosslinks occurs; the extent of rescue of one marker is considerably in excess of that of linked markers.