Rapid deamination of cyclobutane pyrimidine dimer photoproducts at TCG sites in a translationally and rotationally positioned nucleosome in vivo.
Sunlight-induced C to T mutation hot spots in skin cancers occur primarily at methylated CpG sites that coincide with sites of UV-induced cyclobutane pyrimidine dimer (CPD) formation. The C and 5-methyl-C in CPDs are not stable and deaminate to U and T, respectively, which leads to the insertion of A by the DNA damage bypass polymerase η, thereby defining a probable mechanism for the origin of UV-induced C to T mutations. Deamination rates for T(m)CG CPDs have been found to vary 12-fold with rotational position in a nucleosome in vitro. To determine the influence of nucleosome structure on deamination rates in vivo, we determined the deamination rates of CPDs at TCG sites in a stably positioned nucleosome within the FOS promoter in HeLa cells. A procedure for in vivo hydroxyl radical footprinting with Fe-EDTA was developed, and, together with results from a cytosine methylation protection assay, we determined the translational and rotational positions of the TCG sites. Consistent with the in vitro observations, deamination was slower for one CPD located at an intermediate rotational position compared with two other sites located at outside positions, and all were much faster than for CPDs at non-TCG sites. Photoproduct formation was also highly suppressed at one site, possibly due to its interaction with a histone tail. Thus, it was shown that CPDs of TCG sites deaminate the fastest in vivo and that nucleosomes can modulate both their formation and deamination, which could contribute to the UV mutation hot spots and cold spots.
From the Department of Chemistry, Washington University, St. Louis, Missouri 63130.
Vincent J
VJ
Cannistraro
From the Department of Chemistry, Washington University, St. Louis, Missouri 63130.
Santhi
S
Pondugula
From the Department of Chemistry, Washington University, St. Louis, Missouri 63130.
Qian
Q
Song
From the Department of Chemistry, Washington University, St. Louis, Missouri 63130 taylor@wustl.edu.
John-Stephen
JS
Taylor
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D042002:Chromatin Assembly and Disassembly / Q000528:radiation effects; D019175:DNA Methylation / Q000528:radiation effects; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003641:Deamination; D004255:Deoxyribodipyrimidine Photo-Lyase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006367:HeLa Cells; D006657:Histones / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D017665:Hydroxyl Radical / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009707:Nucleosomes / Q000737:chemistry / Q000378:metabolism* / Q000528:radiation effects; D011401:Promoter Regions, Genetic; D011740:Pyrimidine Dimers / Q000737:chemistry* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014466:Ultraviolet Rays; D000099270:Y-Family DNA Polymerases
D001426:Bacterial Proteins; D004259:DNA-Directed DNA Polymerase; D004255:Deoxyribodipyrimidine Photo-Lyase; D006657:Histones; D017665:Hydroxyl Radical; D009707:Nucleosomes; D011740:Pyrimidine Dimers; D011993:Recombinant Fusion Proteins; C112338:Rad30 protein; D000099270:Y-Family DNA Polymerases
5-methylcytosine; DNA damage; chromatin structure; histone; mutagenesis mechanism; nucleosome; photobiology