Synergistic modulation of cyclobutane pyrimidine dimer photoproduct formation and deamination at a TmCG site over a full helical DNA turn in a nucleosome core particle

Abstract

Sunlight-induced C to T mutation hotspots in skin cancers occur primarily at methylated CpG sites that coincide with sites of UV-induced cyclobutane pyrimidine dimer (CPD) formation. The C or 5-methyl-C in CPDs are not stable and deaminate to U and T, respectively, which leads to the insertion of A by DNA polymerase η and defines a probable mechanism for the origin of UV-induced C to T mutations. We have now determined the photoproduct formation and deamination rates for 10 consecutive T=mCG CPDs over a full helical turn at the dyad axis of a nucleosome and find that whereas photoproduct formation and deamination is greatly inhibited for the CPDs closest to the histone surface, it is greatly enhanced for the outermost CPDs. Replacing the G in a T=mCG CPD with A greatly decreased the deamination rate. These results show that rotational position and flanking sequence in a nucleosome can significantly and synergistically modulate CPD formation and deamination that contribute to C to T mutations associated with skin cancer induction and may have influenced the evolution of the human genome.

Rapid Deamination of Cyclobutane Pyrimidine Dimer Photoproducts at TCG Sites in a Translationally and Rotationally Positioned Nucleosome in Vivo

“…Deamination was slower at one site located 73 bases upstream from the dyad center at an intermediate rotational position than at sites located 13 and 24 bases upstream from the dyad at the outermost rotational positions. These results are consistent with what would be predicted based on our previous in vitro study of the effect of rotational position on deamination rate (19). Photoproduct formation was also found to be highly suppressed at an intermediate rotational position 47 bases upstream from the dyad that would be in close proximity to two histone tails, which have been shown to interact with the nucleosome DNA in a crystal structure (25).…”

“…We could, however, determine the deamination rates for the remaining three sites in vitro and in vivo and compare how they changed relative to what we observed previously for the change in deamination rates of T m CG CPDs in a reconstituted nucleosome (19) ( Table 1). Based on their assigned rotational positions, CPDs 3 and 4 would be predicted to deaminate 2.5 times faster relative to CPD 1 when in the nucleosome than when in free DNA (19). The predicted change in deamination rate is within experimental error to the observed change of 1.4 Ϯ 0.9 and 1.7 Ϯ 0.9 for CPDs 3 and 4, respectively ( Table 1).…”

“…We attributed this decrease to a possible interaction between amino acids in the H2A tail and minor groove of the DNA based on analysis of a high resolution nucleosome crystal structure. Although it has been well established that nucleosomes enhance and inhibit the yield of DNA photoproducts according to rotational position (17,19), additional variations with translational position have been observed in a phased nucleosome with defined sequences (57), but the origin of these variations was not determined.…”

“…A 5Ј-flanking G further accelerates the deamination of TC and T m C in duplex DNA over 10-fold compared with other sequence contexts, dropping the half-life to the order of hours (13). Nucleosomes modulate the deamination rate of T m CG CPDs in vitro by up to 50-fold faster for one facing outside compared with one facing inside (5,19). In vivo, a rapidly deaminating CPD would be expected to be more mutagenic because there would be a higher probability that polymerase would encounter a deaminated CPD prior to repair.…”

“…Nucleosomes have been shown to enhance CPD formation at rotational sites furthest away from the histone surface, which was suggested to be due to the greater mobility of the bases at these sites (17,18). In a recent study, we determined the deamination rates for T m CG CPDs at 10 consecutive rotational positions in a nucleosome at the dyad and found a 12-fold difference in rates (19). The maximum rate was with the phosphodiester backbone of the C at the outermost position, and the slowest was with the C at the innermost position.…”