Summary
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 longwave 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.
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Biswal, N., Kleinschmidt, A.K., Spatz, H.C., Trautner, T.A.: Physical properties of the DNA of bacteriophage SP50. Mol. Gen. Genet. 100, 39–55 (1967)
Bron, S., Venema, G.: Ultraviolet inactivation and excision-repair in Bacillus subtilis. I. Construction and characterization of a transformable eightfold auxotrophic strains and two ultraviolet-sensitive derivatives. Mutat. Res. 15, 1–10 (1972)
Buitenwerf, J., Venema, G.: Transformation in Bacillus subtilis. Fate of transforming DNA in transformation deficient mutants. Mol. Gen. Genet. 51, 203–213 (1977a)
Buitenwerf, J., Venema, G.: Transformation in Bacillus subtilis. Biological and physical evidence for a novel DNA-intermediate in synchronously transforming cells. Mol. Gen. Genet. 156, 145–155 (1977b)
Canosi, U., Morelli, G., Trautner, T.A.: The relationship between molecular structure and transformation efficiency of some S. aureus plasmids isolated from B. subtilis. Mol. Gen. Genet. 166, 259–267 (1978)
Cole, R.S.: Psoralen monoadducts and interstrand crosslinks in DNA. Biochim. Biophys. Acta 254, 30–39 (1971)
Cole, R.S., Levitan, D., Swiden, R.: Removal of psoralen interstrand crosslinks from DNA of Escherichia coli: mechanism and genetic control. J. Mol. Biol. 103 39–59 (1976)
Dall'Acqua, F., Rodighiero, G.: The dark interaction between furocoumarins and nucleic acids. Atti. Acad. Nat. Lincei. Cl. Sci. Fis. Mat. Natur. Rend. 40, 412–422 (1966)
Ehrlich, S.D.: Replication and expression of plasmids from Staphylococcus aureus in Bacillus subtilis. Proc. Natl. Acad. Sci. USA 74, 1680–1682 (1977)
Farid, S., Krauch, C.H.: Photochemical and biological reactions of the furocoumarins. In: Radiat. Res. Proc. Third Int. Congr. Radiat. Res. 1966 (G. Silini ed.) pp. 869–886. Amsterdam: North-Holland 1967
Iordanescu, S.: Recombinant plasmid obtained from two different compatible staphylococcal plasmids. J. Bacteriol. 124, 597–601 (1975)
Joenje, H., Venema, G.: Different nuclease activities in competent and noncompetent Bacillus subtilis. J. Bacteriol. 122, 25–33 (1975)
Kirby, K.S.: A new method for the isolation of deoxyribonucleic acid: evidence on the nature of bonds between deoxyribonucleic acids and protein. Biochem. J. 66, 495–504 (1957)
Krauch, G.H., Krämer, D.M., Wacker, A.: Zum Wirkungsmechanismus photodynamischer Furocoumarine, photoreaktion von [4-14C] psoralen mit DNA, RNS, homopolynucleotide und nucleosiden. Photochem. Photobiol. 6, 341–354 (1967)
Lacks, S.: Molecular fate of DNA in genetic transformation of pneumococcus. J. Mol. Biol. 5, 119–131 (1962)
Lacks, S., Greenberg, B., Neuberger, M.: Role of a deoxyribonuclease in the genetic transformation of Diplococcus pneumoniae. Proc. Natl. Acad. Sci. USA 71, 2305–2309 (1976)
Loveday, K.S., Fox, M.S.: Fate of transfecting DNA in Bacillus subtilis. Virology 85, 387–403
Morelli, G., Fisseau, C., Behrens, B., Trautner, T.A., Luh, J., Ratcliff, S.W., Allison, D.P., Ganesan, A.T.: The genome of B. subtilis phage SPP1: the topology of DNA molecules. Mol. Gen. Genet. 168, 153–164 (1979)
Morrison, D.A., Guild, W.R.: Transformation and deoxyribonucleic acid size: extent of degradation on entry varies with size of donor. J. Bacteriol. 112, 1157–1168 (1972)
Musajo, L.: Photochemical interactions between skin photosensitizing furocoumarins and DNA. In: Radiat. Res. Proc. Third Int. Congr. Radiat. Res. 1966 (G. Salini, ed.) pp. 893–912. Amsterdam: North-Holland 1967
Musajo, L., Bordin, F., Bevilacqua, R.: Photoreactions at 3655Å, linking the 3–4 double bond of furocoumarins with pyrimidine basis. Photochem. Photobiol. 6, 927–931 (1967a)
Musajo, L., Bordin, F., Caporal, G., Marciani, S., Ragatti, C.: Photoreactions at 3655 Å between pyrimidine basis and skin photosensitizing furocoumarins. Photochem. Photobiol. 6, 711–719 (1967b)
Piechowska, M., Fox, M.S.: Fate of transforming deoxyribonucleate in Bacillus subtilis. J. Bacteriol. 108, 680–689 (1971)
Rottländer, E., Trautner, T.A.: Genetic and transfection studies with B. subtilis phage SP50. I. Phage mutants with restricted growth on B. subtilis strain 168. Mol. Gen. Genet. 108, 47–60 (1970)
Scott, B.R., Pathak, M.A., Mohn, G.R.: Molecular and genetic basis of furocoumarin reactions. Mutat. Res. 39, 29–74 (1976)
Spatz, H. Ch., Trautner, T.A.: One way to do experiments on gene conversion? Mol. Gen. Genet. 109, 84–106 (1970)
Spatz, H.Ch., Trautner, T.A.: The role of recombination in transfection of B. subtilis. Mol. Gen. Genet. 113, 174–190 (1971)
Spizizen, J.: Transformation of biochemically deficient strains of Bacillus subtilis by deoxyribonucleate. Proc. Natl. Acad. Sci. USA 44, 1072–1078 (1958)
Venema, G., Pritchard, R.H., Venema-Schröder, T.: Fate of transforming deoxyribonucleic acid in Bacillus subtilis. J. Bacteriol. 89, 1250–1255 (1965)
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Venema, G., Canosi, U. The effect of trimethylpsoralen — Crosslinks on entry of donor DNA in transformation and transfection of Bacillus subtilis . Molec. Gen. Genet. 179, 1–11 (1980). https://doi.org/10.1007/BF00268439
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DOI: https://doi.org/10.1007/BF00268439