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Z-DNA-forming sequences are shown to elicit a biological response in Escherichia coli. Plasmids containing sequences capable of adopting the Z conformation (GC and CA/GT) are shown to be hot spots for spontaneous deletions.All the deletions involve an even number of base pairs. The distribution of the deletion events shows that the process ends when the Z-DNA-forming sequence has been reduced to a size no longer capable of adopting the Z conformation at natural superhelical density. Lac-->Lac+ (3n+1) bp insert: the loss of (3p+1) bp or the addition of (3p+2) bp are detected (3n+2) bp insert: the loss of (3p+2) bp or the addition of (3p+ 1) bp are detected Lac + ->Lac -(3n) bp insert: the loss or the addition of (3p+ 1) bp or (3p+2) bp are detected FIG. 1. Strategy for the frameshift mutation assay. Various lengths of alternating purine-pyrimidine bases (such as GC, GT, or AT) have been cloned in the early part of the lacZ gene of plasmid pUC8. As shown, these plasmids were used in the f-galactosidase a complementation assay to monitor the frameshift mutation frequency within the inserted sequence.intensities were used to calculate the frequencies of the different mutational events.Biochemical analysis. About 104 transformants containing plasmid pUC-(GC)13 were pooled, and their plasmid DNA was analyzed on sequencing gels as described for the phenotypic analysis. The autoradiogram revealed a high intensity band of starting plasmid and several lighter bands. We have only quantified the bands corresponding to the "long deletion events." Two-Dimensional Agarose Gel Technologies. The topoisomers were run on a 2% agarose gel, 10 cm long, in TAE buffer (30). The first direction was run without intercalating agent at 3 V/cm for 17 hr. In the second direction, the gel (turned 900) Abbreviations: SADE, small addition/deletion event; LDE, long deletion event; AAF, 2-acetylaminofluorene. 7465The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

ColE1 DNA has been allowed to react in vitro with N-acetoxy-N-2-[14C]acetylaminofluorene in the range of 0-15 N-2-[14C]acetylaminofluorene residues bound per molecule of DNA, at the C8 of guanine residues. Purified rabbit antibodies to both N-2-(guanosine-8-yl)-acetylaminofluorene and native DNA that had reacted with N-acetoxy-N-2-acetylaminofluorene were shown by electron microscopy to recognize specifically the acetylaminofluorene-modified ColE1 DNA. The antibodies bound to DNA were visualized either per se or after reaction with goat anti-rabbit immunoglobulins coupled with ferritin. There was a linear relationship between the average number of antibodies bound per DNA molecule and the number of N-2-(deoxyguanosine-8yl)-acetylaminofluorene residues per DNA molecule. The slope of this straight line was equal to 0.4. Due to the bivalence of the immunoglobulins one would expect a value of 0.5; we actually observed an important fraction of the bound antibodies crosslinking two parts of the same (or of another) DNA molecule.

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By means of an immunoradiometric assay the induction of protein RecA in E. coli PQ 10 was measured after treatment by two related carcinogens. On an adduct basis N-Acetoxy-N-2-acetylaminofluorene was shown to induce the protein RecA at a similar level as U.V. On the other hand, N-hydroxy-N-2-aminofluorene shows only a poor induction capacity of the RecA protein. The difference in the SOS inducing potential of the aminofluorene and acetylaminofluorene adducts is discussed in relation to the major difference in the local conformational change the two adducts induce in DNA.