Modulation of an ultraviolet mutational hotspot in a shuttle vector in Xeroderma cells

Abstract

Ultraviolet mutagenesis of the shuttle vector plasmid pZ189 in Xeroderma Pigmentosum cells yields a mutational pattern marked by hotspots at photoproduct sites on both strands of the supF marker gene. In order to test the influence of strand orientation on the appearance of hotspots the mutagenesis study was repeated on a vector with the supF gene in the inverted orientation. We recovered a pattern the same as that in the earlier work and conclude that the nature of the DNA polymerase involved in the replication of specific strands is not a primary determinant of hotspot occurrence in this system. One of the hotspots lies in an 8 base palindrome while the corresponding site on the other strand was not a hotspot. These results were obtained with calcium phosphate transfection of the UV treated vector. When DEAE dextran was used as a transfection agent both sites in the palindrome were hotspots. In a mixing experiment the calcium phosphate pattern was recovered. Our data suggest that the sequence determinants of mutational probability at these two sites lie outside the 8 bases of the palindrome and that mutagenesis at one, but not the other, site is sensitive to perturbation of cellular calcium levels.