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Single nucleotide positions have proximal and distal influence on UV mutation hotspots and coldspots.

Base substitution mutation frequency is influenced by the sequence context surrounding lesions in the DNA. We have been studying ultraviolet mutagenesis in human repair-deficient cells in the supF marker gene carried in a shuttle vector plasmid. There are prominent hotspots, on opposite strands, at the 5' TC sites in the eight base palindrome 5' CTTCGAAG. Recently, we developed a reporter system which permits sequence manipulation in the vicinity of mutational hotspots. We have used the 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 the 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 modulation of hotspot activity at other sites as a function of single base changes as much as 80 bases away from the hotspots. In these instances, the site of the changed base was in the unreplicated template ahead of the primer terminus when the polymerase encountered the relevant photoproduct. Our results indicate that sequence context has both proximal and distal consequences for mutagenesis.