Photoproduct frequency is not the major determinant of UV base substitution hot spots or cold spots in human cells.

Abstract

The role of UV radiation-induced photoproducts in initiating base substitution mutations in human cells was examined by measuring photoproduct frequency distributions and mutations in a supF tRNA gene on a shuttle vector plasmid transfected into DNA repair-deficient cells (xeroderma pigmentosum, complementation group A) and into normal cells. Frequencies of cyclobutane dimers and pyrimidine-pyrimidone (6-4) photoproducts varied by as much as 80-fold at different dipyrimidine sites within the gene. AU transition mutations occurred at dipyrimidine sites, predominantly at cytosine, with a 17-fold variation in mutation frequency between different sites. Removal of >99% of the cyclobutane dimers by in vitro photoreactivation before transfection reduced the mutation frequency while preserving the mutation distribution, indicating that (i) cytosine-containing cyclobutane dimers were the major mutagenic lesions at these sites and (ii) cytosinecontaining non-cyclobutane dimer photoproducts were also mutagenic lesions. However, at individual dipyrimidine sites neither the frequency of cyclobutane dimers nor the frequency of pyrimidine-pyrimidone (6-4) photoproducts correlated with the mutation frequency, even in the absence of excision repair. Mutation hot spots occurred at sites with low or high frequency of photoproduct formation and mutation cold spots occurred at sites with many photoproducts. These results suggest that although photoproducts are required for UV mutagenesis, the prominence of most mutation hot spots and cold spots is primarily determined by DNA structural features rather than by the frequency of DNA photoproducts.

The specificity of UV-induced mutations at an endogenous locus in mammalian cells.

“…Brash et al (39) have recently shown that in mammalian cells UV-induced mutational hot spots do not correlate with the frequency of photoproduct formation. Furthermore, repair processes may be of considerable importance since region-to-region and site-to-site variations in repair (40,41) and the fidelity of DNA polymerase (42) have been observed.…”

DNA strand specificity for UV-induced mutations in mammalian cells.

“…The two types of DNA damage which are considered to be primarily responsible for the lethal and mutagenic effects of UV irradiation are the cyclobutane pyrimidine dimer (7,19) and the (6-4) pyrimidine-pyrimidone photoproduct (3, 10). Still, controversy exists over the relative importance of both types of lesions for cell killing and mutagenesis (4,6,13,16).It has been shown for cultured Chinese hamster and human cells that removal of dimers from the transcriptionally active dhfr gene is more rapid than for the genome overall (1, 2, 11) and that possibly even a strand specificity in DNA repair exists (12). Dimers were removed at a faster rate in human cells and to a larger extent in Chinese hamster ovary (CHO) cells from the transcribed strand from the amplified dhfr gene than from the nontranscribed strand.…”

“…The two types of DNA damage which are considered to be primarily responsible for the lethal and mutagenic effects of UV irradiation are the cyclobutane pyrimidine dimer (7,19) and the (6-4) pyrimidine-pyrimidone photoproduct (3, 10). Still, controversy exists over the relative importance of both types of lesions for cell killing and mutagenesis (4,6,13,16).…”

DNA sequence context affects UV-induced mutagenesis in Escherichia coli

“…For example, the frequencies of CAA and AAA mutations are much greater at the 5′-TT site than at the 5′-CT site (Table 3, compare rows 1 and 2). This might indicate a greater capacity for DNA damage, since TTCPDs occur more often than CT-CPDs, combined with the knowledge that products can form at TT sites, but are unlikely at 5′-CT sites [28]. The increase in the proportion of CAA mutations is consistent with the specificity of the TT product (Table 1).…”

“…Therefore, 5′-CT dimers may present a different obstacle for DNA polymerase, altering the base selection process. Since products probably do not readily form at 5′-CT sequences [7,28], the premutagenic lesion is likely a CPD.…”