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Bohm, Kaitlynne A.; Sivapragasam, Smitha; Wyrick, John J.
STAR protocols, 03/2022, Letnik: 3, Številka: 1Journal Article
Exposure to ultraviolet (UV) light induces DNA damage, predominantly cyclobutane pyrimidine dimers (CPD) and 6,4-photoproducts (6,4-PP), as well as rare, atypical photoproducts at thymidine-adenine (TA) sequences. We have recently shown ‘TA’ photoproducts are induced in UV-irradiated oligonucleotides and across the budding yeast genome. Here, we describe a protocol for mapping atypical ‘TA’ photoproducts in vitro and in vivo. This protocol overcomes the technical challenges involved in accurately mapping such rare photoproducts by using ultraviolet damage endonuclease (UVDE) enzymes. For complete details on the use and execution of this protocol, please refer to Laughery et al. (2020). Display omitted •UVDE enzyme cleaves ‘TA’ photoproducts in UV-irradiated oligos and the yeast genome•CPD photolyase treatment ensures only 6,4-PPs and atypical lesions are mapped•Bioinformatic pipelines precisely locate where these ‘TA’ lesions formed in vivo Exposure to ultraviolet (UV) light induces DNA damage, predominantly cyclobutane pyrimidine dimers (CPD) and 6,4-photoproducts (6,4-PP), as well as rare, atypical photoproducts at thymidine-adenine (TA) sequences. We have recently shown ‘TA’ photoproducts are induced in UV-irradiated oligonucleotides and across the budding yeast genome. Here, we describe a protocol for mapping atypical ‘TA’ photoproducts in vitro and in vivo. This protocol overcomes the technical challenges involved in accurately mapping such rare photoproducts by using ultraviolet damage endonuclease (UVDE) enzymes.
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