Pyrimidine (6-4) pyrimidone photoproduct mapping after sublethal UVC doses: nucleotide resolution using terminal transferase-dependent PCR.
UVC irradiation of genomic DNA induces two main types of potentially mutagenic base modifications: cyclobutane pyrimidine dimers (CPDs) and the less frequent (15-30% of CPD levels) pyrimidine (6-4) pyrimidone photoproducts (6-4PP). Ligation-mediated PCR (LMPCR), a genomic sequencing technique, allows CPD mapping at nucleotide resolution following irradiation with sublethal doses of UVB or UVC for most cell types. In contrast, a dose of 80 J/m(2) of UVC that is lethal for the majority of cell types is necessary to map 6-4PP by the LMPCR technique. This compromises the use of LMPCR to study the repair of 6-4PP. To date, no other techniques have been developed to study 6-4PP repair at nucleotide resolution. We have therefore adapted a recently developed technique for the mapping of 6-4PP: terminal transferase-dependent PCR (TDPCR). TDPCR is in many ways similar to LMPCR. This technique is more sensitive and allows the mapping of 6-4PP at UVC doses as low as 10 J/m(2) in genomic DNA and in living cells.
Rochette
Patrick J
PJ
Division of Pathology, Department of Medical Biology, Université Laval, Quebec, PQ, Canada.
Bastien
Nathalie
N
Todo
Takeshi
T
Drouin
Régen
R
D001665:Binding Sites; D004247:DNA / Q000528:radiation effects; D005347:Fibroblasts / Q000528:radiation effects; D006801:Humans; D009841:Oligonucleotides / Q000737:chemistry / Q000528:radiation effects*; D011743:Pyrimidines / Q000737:chemistry / Q000528:radiation effects*; D011744:Pyrimidinones / Q000737:chemistry / Q000528:radiation effects*; D014466:Ultraviolet Rays
D009841:Oligonucleotides; D011743:Pyrimidines; D011744:Pyrimidinones; D004247:DNA; C030986:pyrimidine