SINGLE NUCLEOTIDE POSITIONS HAVE PROXIMAL AND DISTAL INFLUENCE ON UV MUTATION HOTSPOTS AND COLDSPOTS

Base substitution mutation frequency is influenced by the sequence con text surrounding lesions in the DNA. We have been studying ultraviolet mutagenesis in human repair-deficient cells in the supF marker gene c arried in a shuttle vector plasmid. There are prominent hotspots, on o pposite strands, at the 5' TC sites in the eight base palindrome 5' CT TCGAAG. Recently, we developed a reporter system which permits sequenc e manipulation in the vicinity of mutational hotspots. We have used th e system to characterize the influence of individual positions in the palindrome on the frequency of mutagenesis at the two UV hotspots. In this paper we have determined the contribution of bases at the second and third positions in the palindrome. Changes in bases that were in t he primer template duplex when the replication complex encountered the photoproducts at one of the hotspot sites significantly increased or decreased the probability of mutations at the site. We also observed m odulation of hotspot activity at other sites as a function of single b ase changes as much as 80 bases away from the hotspots. In these insta nces, the site of the changed base was in the unreplicated template ah ead of the primer terminus when the polymerase encountered the relevan t photoproduct. Our results indicate that sequence context has both pr oximal and distal consequences for mutagenesis. (C) 1996 Academic Pres s Limited