Published online 19 May 2017
Nucleic Acids Research, 2017, Vol. 45, No. 12 7031–7041 doi: 10.1093/nar/gkx427
Modulation of cyclobutane thymine photodimer formation in T11-tracts in rotationally phased nucleosome core particles and DNA minicircles Kesai Wang and John-Stephen A. Taylor* Department of Chemistry, Washington University, St Louis, MO 63130, USA Received February 03, 2017; Revised May 01, 2017; Editorial Decision May 02, 2017; Accepted May 11, 2017
ABSTRACT Cyclobutane pyrimidine dimers (CPDs) are DNA photoproducts linked to skin cancer, whose mutagenicity depends in part on their frequency of formation and deamination. Nucleosomes modulate CPD formation, favoring outside facing sites and disfavoring inward facing sites. A similar pattern of CPD formation in protein-free DNA loops suggests that DNA bending causes the modulation in nucleosomes. To systematically study the cause and effect of nucleosome structure on CPD formation and deamination, we have developed a circular permutation synthesis strategy for positioning a target sequence at different superhelix locations (SHLs) across a nucleosome in which the DNA has been rotationally phased with respect to the histone octamer by TG motifs. We have used this system to show that the nucleosome dramatically modulates CPD formation in a T11 -tract that covers one full turn of the nucleosome helix at seven different SHLs, and that the position of maximum CPD formation at all locations is shifted to the 5 -side of that found in mixed-sequence nucleosomes. We also show that an 80-mer minicircle DNA using the same TG-motifs faithfully reproduces the CPD pattern in the nucleosome, indicating that it is a good model for protein-free rotationally phased bent DNA of the same curvature as in a nucleosome, and that bending is modulating CPD formation. INTRODUCTION It is well established that DNA photoproducts produced by sunlight are responsible for the majority of the mutations associated with skin cancers (1–4). What is not so well established is the physical or mechanistic origin of the variation in mutation type and frequency within a gene, which must result from a complex interplay between the frequency of photoproduct formation, chemical transformation, re* To
pair and translesion synthesis. To begin to dissect the various contributions of these factors, we have been examining the role of chromatin structure on DNA photoproduct formation and subsequent deamination. It was demonstrated many decades ago that nucleosomes can modulate cyclobutane pyrimidine dimer (CPD) formation in mixed sequence genomic DNA with a 10–11 bp periodicity that was attributed to the effect of bending on the conformation and dynamics of DNA (5,6). A similar effect was observed to occur in a protein-free DNA loop which supported the idea that bending, and not protein DNA interactions were the primary cause of the modulation of photoproduct formation (7). The original studies of CPD modulation by nucleosomes were carried out with nucleosomes containing mixed sequence DNA obtained from the degradation of chromatin. Such sequences may be biased to bend around the nucleosome and obscure the relationship between sequence, rotational position and CPD formation. In a study of 254 nm induced CPD formation in nucleosome bound 5S rRNA gene with a defined sequence, a
Nucleic Acids Research, 2017, Vol. 45, No. 12 7031–7041 doi: 10.1093/nar/gkx427
Modulation of cyclobutane thymine photodimer formation in T11-tracts in rotationally phased nucleosome core particles and DNA minicircles Kesai Wang and John-Stephen A. Taylor* Department of Chemistry, Washington University, St Louis, MO 63130, USA Received February 03, 2017; Revised May 01, 2017; Editorial Decision May 02, 2017; Accepted May 11, 2017
ABSTRACT Cyclobutane pyrimidine dimers (CPDs) are DNA photoproducts linked to skin cancer, whose mutagenicity depends in part on their frequency of formation and deamination. Nucleosomes modulate CPD formation, favoring outside facing sites and disfavoring inward facing sites. A similar pattern of CPD formation in protein-free DNA loops suggests that DNA bending causes the modulation in nucleosomes. To systematically study the cause and effect of nucleosome structure on CPD formation and deamination, we have developed a circular permutation synthesis strategy for positioning a target sequence at different superhelix locations (SHLs) across a nucleosome in which the DNA has been rotationally phased with respect to the histone octamer by TG motifs. We have used this system to show that the nucleosome dramatically modulates CPD formation in a T11 -tract that covers one full turn of the nucleosome helix at seven different SHLs, and that the position of maximum CPD formation at all locations is shifted to the 5 -side of that found in mixed-sequence nucleosomes. We also show that an 80-mer minicircle DNA using the same TG-motifs faithfully reproduces the CPD pattern in the nucleosome, indicating that it is a good model for protein-free rotationally phased bent DNA of the same curvature as in a nucleosome, and that bending is modulating CPD formation. INTRODUCTION It is well established that DNA photoproducts produced by sunlight are responsible for the majority of the mutations associated with skin cancers (1–4). What is not so well established is the physical or mechanistic origin of the variation in mutation type and frequency within a gene, which must result from a complex interplay between the frequency of photoproduct formation, chemical transformation, re* To
pair and translesion synthesis. To begin to dissect the various contributions of these factors, we have been examining the role of chromatin structure on DNA photoproduct formation and subsequent deamination. It was demonstrated many decades ago that nucleosomes can modulate cyclobutane pyrimidine dimer (CPD) formation in mixed sequence genomic DNA with a 10–11 bp periodicity that was attributed to the effect of bending on the conformation and dynamics of DNA (5,6). A similar effect was observed to occur in a protein-free DNA loop which supported the idea that bending, and not protein DNA interactions were the primary cause of the modulation of photoproduct formation (7). The original studies of CPD modulation by nucleosomes were carried out with nucleosomes containing mixed sequence DNA obtained from the degradation of chromatin. Such sequences may be biased to bend around the nucleosome and obscure the relationship between sequence, rotational position and CPD formation. In a study of 254 nm induced CPD formation in nucleosome bound 5S rRNA gene with a defined sequence, a
