Rochette Patrick J, Bastien Nathalie, Todo Takeshi, Drouin Régen
Division of Pathology, Department of Medical Biology, Université Laval, Quebec, PQ, Canada.
Photochem Photobiol. 2006 Sep-Oct;82(5):1370-6. doi: 10.1562/2004-12-01-RA-390.
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.
基因组DNA的紫外线C（UVC）照射会诱导两种主要类型的潜在诱变碱基修饰：环丁烷嘧啶二聚体（CPD）和频率较低的（CPD水平的15 - 30%）嘧啶（6 - 4）嘧啶酮光产物（6 - 4PP）。连接介导的PCR（LMPCR）是一种基因组测序技术，对于大多数细胞类型，在用亚致死剂量的UVB或UVC照射后，它能以核苷酸分辨率进行CPD图谱分析。相比之下，要用LMPCR技术对6 - 4PP进行图谱分析，需要80 J/m²的UVC剂量，而这个剂量对大多数细胞类型是致死的。这就限制了LMPCR用于研究6 - 4PP修复的应用。到目前为止，尚未开发出其他技术以核苷酸分辨率研究6 - 4PP修复。因此，我们采用了一种最近开发的用于6 - 4PP图谱分析的技术：末端转移酶依赖性PCR（TDPCR）。TDPCR在很多方面与LMPCR相似。该技术更灵敏，能在低至10 J/m²的UVC剂量下对基因组DNA和活细胞中的6 - 4PP进行图谱分析。
