Takasawa Kohei, Masutani Chikahide, Hanaoka Fumio, Iwai Shigenori
Division of Chemistry, Graduate School of Engineering Science, Osaka University, 1-3 Machikaneyama, Toyonaka, Osaka 560-8531, Japan.
Nucleic Acids Res. 2004 Mar 12;32(5):1738-45. doi: 10.1093/nar/gkh342. Print 2004.
The cytosine base in DNA undergoes hydrolytic deamination at a considerable rate when UV radiation induces formation of a cyclobutane pyrimidine dimer (CPD) with an adjacent pyrimidine base. We have synthesized a phosphoramidite building block of a cis-syn cyclobutane thymine-uracil dimer (T[]U), which is the deaminated form of the CPD at a TC site, and incorporated it into oligodeoxyribonucleotides. The previously reported method for synthesis of the thymine dimer (T[]T) was applied, using partially protected thymidylyl-(3'-5')-2'-deoxyuridine as the starting material, and after triplet- sensitized irradiation, the configuration of the base moiety in the major product was determined by NMR spectroscopy. Presence of the cis-syn cyclobutane dimer in the obtained oligonucleotides was confirmed by UV photoreversal and reaction with T4 endonuclease V. Using a 30mer containing T[]U, translesion synthesis by human DNA polymerase eta was analyzed. There was no difference in the results between the templates containing T[]T and T[]U and pol eta bypassed both lesions with the same efficiency, incorporating two adenylates. This enzyme showed fidelity to base pair formation, but this replication causes a C-->T transition because the original sequence is TC.
当紫外线辐射诱导与相邻嘧啶碱基形成环丁烷嘧啶二聚体（CPD）时，DNA中的胞嘧啶碱基会以相当高的速率发生水解脱氨反应。我们合成了顺式-顺式环丁烷胸腺嘧啶-尿嘧啶二聚体（T[]U）的亚磷酰胺砌块，它是TC位点处CPD的脱氨形式，并将其掺入寡脱氧核糖核苷酸中。采用先前报道的胸腺嘧啶二聚体（T[]T）合成方法，以部分保护的胸苷酰基-（3'-5'）-2'-脱氧尿苷为起始原料，经过三重态敏化照射后，通过核磁共振光谱法确定主要产物中碱基部分的构型。通过紫外光逆转和与T4内切核酸酶V反应，证实了所得寡核苷酸中存在顺式-顺式环丁烷二聚体。使用含有T[]U的30聚体分析了人DNA聚合酶η的跨损伤合成。含有T[]T和T[]U的模板之间的结果没有差异，聚合酶η以相同的效率绕过这两种损伤，掺入两个腺苷酸。这种酶对碱基对形成具有保真度，但这种复制会导致C→T转换，因为原始序列是TC。
