O'Day C L, Burgers P M, Taylor J S
Department of Chemistry, Washington University, St Louis, MO 63130.
Nucleic Acids Res. 1992 Oct 25;20(20):5403-6. doi: 10.1093/nar/20.20.5403.
Calf thymus proliferating cell nuclear antigen (PCNA) promoted DNA synthesis past cis-syn and trans-syn-I cyclobutane thymine dimers by calf thymus DNA polymerase delta (Pol delta) in vitro. Templates containing site-specific cis-syn and trans-syn-I thymine dimers were prepared via a combination of solid phase synthesis with photoproduct building blocks and DNA ligation. Extension of a 15-mer primer on the UV dimer-containing templates by Pol delta produced termination and bypass products in a dNTP and PCNA dependent manner. In the absence of PCNA and at dNTP concentrations varying between 1 and 100 microM, Pol delta could not bypass the cis-syn dimer and terminated elongation one nucleotide prior to the 3'-T of the dimer. DNA synthesis past the trans-syn-I dimer was even less efficient. In the presence of PCNA, termination occurred primarily one nucleotide prior to the 3'-T of both dimers at 1 microM dNTPs but opposite the 5'-T of the dimers at 100 microM dNTPs. In addition, under the latter conditions, bypass of the dimers was observed, to the extent of about 30% of the products for the cis-syn dimer and about 15% for the trans-syn-I dimer.
小牛胸腺增殖细胞核抗原（PCNA）在体外通过小牛胸腺DNA聚合酶δ（Pol δ）促进DNA合成越过顺式-顺式和反式-顺式-I环丁烷胸腺嘧啶二聚体。通过固相合成与光产物构建模块及DNA连接相结合的方法，制备了含有位点特异性顺式-顺式和反式-顺式-I胸腺嘧啶二聚体的模板。Pol δ在含紫外线二聚体的模板上对15聚体引物的延伸以依赖dNTP和PCNA的方式产生终止和绕过产物。在没有PCNA且dNTP浓度在1至100微摩尔之间变化时，Pol δ无法绕过顺式-顺式二聚体，并在二聚体3'-T之前一个核苷酸处终止延伸。越过反式-顺式-I二聚体的DNA合成效率更低。在有PCNA存在的情况下，在1微摩尔dNTP时，终止主要发生在两个二聚体3'-T之前一个核苷酸处，但在100微摩尔dNTP时，终止发生在二聚体5'-T相对处。此外，在后一种条件下，观察到二聚体的绕过现象，顺式-顺式二聚体的绕过产物约占30%，反式-顺式-I二聚体的绕过产物约占15%。
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