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Electrophoretic mobility of PM2 DNA treated with ultimate chemical carcinogens or with ultraviolet light

Elektrophoretische Beweglichkeit von PM2 DNA nach Umsetzung mit direkt wirkenden chemischen Carcinogenen oder nach Bestrahlung mit ultraviolettem Licht

  • Original Papers
  • Experimental Oncology
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Zusammenfassung

Die superhelikale DNA des Pseudomonas Phagen PM2 wurde mit Ultraviolettlicht bestrahlt oder mit kovalent bindenden Carcinogenen wie 7-Brommethyl-benz[a]anthrazen, (Ac)2 ONFln, K-Region Expoxiden und Alkylantien umgesetzt. Mittels Gelelektrophorese wurde die Wanderungsgeschwindigkeit der DNA-Produkte bestimmt. In Trenngelen mit einem Agarosegehalt von 1,3–1,9% nahm die Wanderungsgeschwindigkeit der DNA mit steigender Modifikationsdichte (steigenden Carcinogen-Konzentrationen bzw. UV-Dosen) ab. Dieses Phänomen ist wahrscheinlich darauf zurückführen, daß die DNA-Modifikation eine Abnahme der Superhelix-Dichte bewirkte, im Zuge derer das ursprünglich kompakte, geknäuelte DNA-Molekül schließlich eine offen-zirkuläre Konformation annahm. Ein Vergleich der DNA-Modifikationsdichte mit der damit verbundenen Abnahme der Wanderungsgeschwindigkeit machte deutlich, daß der Grad der Superhelizität sehr empfindlich mit den chemischen DNA-Veränderungen variierte.

DNA-Proben, die in 1.6%igen Agarosegelen eine Wanderungsverzögerung bis zu 70% der Kontroll-DNA zeigten, liefen in 0.4%igen Trenngelen merkwürdigerweise schneller als die Kontrollen. Somit ist in Trenngelen von einem 1,3–1,9igen Agarosegehalt die Abnahme der Superhelix-Dichte von einer Zunahme des Reibungskoeffizienten begleitet; dagegen scheint in 0,4–0,9%igen Agarosegelen dieselbe Abnahme der Superhelix-Dichte zu einer höheren Flexibilität des Makromoleküls und/oder der Freisetzung zusätzlicher elektrischer Ladungen zu führen.

Summary

Superhelical DNA of the Pseudomonas phage PM2 was irradiated with UV-light or reacted with covalently binding carcinogens, such as 7-bromomethyl-benz[a]anthracene, (Ac)2ONFln, K-region epoxides, and alkylating agents. Migration velocity of the DNA products was determined using agarose gel electrophoresis. In gels of more than 1.3%–1.9% agarose, modified PM2 DNA exhibited a dose-(concentration-)dependent decrease of migration velocity. This phenomenon is probably due to a decrease in superhelix density which caused the compact DNA coil to assume eventually an open-circular conformation. Comparison of the extent of DNA modification with the decrease of migration velocity revealed that the superhelical structure sensitively reflected the chemical DNA alterations.

DNA species exhibiting, in 1.6% agarose gels, a migration velocity of up to 30% of that of control DNA showed an increase of velocity in 0.4% agarose. Therefore, in 1.3%–1.9% agarose gels, the decrease of superhelix density is accompanied by an increase of the frictional coefficient, whereas in 0.4%–0.9% agarose gels the same decrease of superhelix density apparently led to a higher degree of flexibility of the macromolecule and/or exposure of additional electric charges.

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Abbreviations

(Ac)2ONFln:

N-acetoxy-2-acetylaminofluorene

(Ac)NHFln:

2-acetylaminofluorene

MeSO2OMe:

methyl methanesulfonate

PM2 DNA (form I):

superhelical DNA of the Pseudomonas phage PM2

PM2 DNA:

nicked form, the open-stranded DNA duplex of PM2

UV-light:

ultraviolet light

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Authors and Affiliations

  1. Institut für Biochemie, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280, D-6900, Heidelberg, Federal Republic of Germany

    H. W. Thielmann & R. Hecht

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  1. H. W. Thielmann
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  2. R. Hecht
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Additional information

This work was supported by the Deutsche Forschungsgemeinschaft, SFB 136. The authors wish to thank Mrs. L. Berry for critically reading the manuscript and for helpful suggestions

This work includes part of the doctoral thesis of R. Hecht, Naturwissenschaftliche Gesamtfakultät der Universität Heidelberg, 1978

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Thielmann, H.W., Hecht, R. Electrophoretic mobility of PM2 DNA treated with ultimate chemical carcinogens or with ultraviolet light. J Cancer Res Clin Oncol 96, 243–257 (1980). https://doi.org/10.1007/BF00408097

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  • DOI: https://doi.org/10.1007/BF00408097

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