Genetics, Vol 120, 863-873, Copyright © 1988

INVESTIGATIONS

Mechanisms of Mutagenesis by a Bulky DNA Lesion at the Guanine N7 Position

K. Sambamurti, J. Callahan, X. Luo, C. P. Perkins, J. S. Jacobsen and M. Z. Humayun
Department of Microbiology and Molecular Genetics, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, New Jersey 07103

In order to examine the mechanisms of mutagenesis by a bulky DNA lesion at the guanine N7 position, the replicative form DNA of phage M13AB28 (mp8 without the amber codons in phage genes) was modified in vitro with aflatoxin B(1)-2,3-dichloride and transfected into appropriate Escherichia coli cells. Forward mutations in the lacZ {alpha}-complementing gene segment were identified as light blue or colorless plaques on appropriate indicator plates, isolated, and defined by DNA sequencing. Transfection of modified DNA into uvrA(-)/mucAB(+) cells without prior UV (SOS) induction increased mutation frequency eight-fold over untreated DNA, whereas this increase was 12-fold upon SOS induction. Transfection of modified DNA after conversion of the primary guanine-aflatoxin lesions to the stable imidazole ring-opened formamidopyrimidine-aflatoxin suggested that these lesions were nearly equally mutagenic. A majority of point mutations under all conditions affected G:C bp. Base substitutions were in the majority, but significant frameshift mutagenesis was also detected in SOS-induced cells. Both G-to-T transversions and G-to-A transitions were produced at equal efficiency and together accounted for virtually all of the base substitutions induced by the primary lesions. Point mutations occurred predominantly at predicted damage hotspots. The characteristics of base substitution and frameshift mutations, together with available information point to multiple mechanisms of mutagenesis by this class of mutagens. The data indicate that primary lesions have the properties of both a noninstructional and pseudo-instructional lesion. In addition, the sequence context appears to play a role in determining whether a frameshift or a base substitution is induced by this bulky lesion.

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