Clark J M
Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709.
Gene. 1991 Jul 31;104(1):75-80. doi: 10.1016/0378-1119(91)90467-p.
DNA polymerases normally catalyze DNA synthesis in a template-directed manner. Generally, the continuity of the phosphodiester backbone of the template strand was thought to be an absolute requirement for DNA synthesis. Here, I demonstrate that a 3'-exonuclease-deficient derivative of the Klenow (large) fragment of Escherichia coli DNA polymerase I (PolIk) can carry out DNA synthesis on discontinuous templates in vitro. Addition of multiple nucleotides (nt) to the 3' end of a blunt-end duplex, templated by unlinked single-stranded oligodeoxyribonucleotides (oligos), was monitored electrophoretically. The reaction was demonstrable with either homopolymers or mixed-sequence oligos, but showed a requirement for complementarity between the first nt added to the duplex and the 3' nt of the unlinked oligo. These results demonstrate that continuity of the phosphodiester backbone of the template strand is not absolutely required for in vitro DNA synthesis by a 3'-exonuclease-deficient form of PolIk.
DNA聚合酶通常以模板指导的方式催化DNA合成。一般来说，模板链磷酸二酯主链的连续性被认为是DNA合成的绝对必要条件。在此，我证明了大肠杆菌DNA聚合酶I（PolIk）的Klenow（大片段）的3'-核酸外切酶缺陷衍生物能够在体外不连续模板上进行DNA合成。通过电泳监测了由未连接的单链寡脱氧核糖核苷酸（寡核苷酸）作为模板，在平端双链体的3'末端添加多个核苷酸（nt）的情况。该反应在同聚物或混合序列寡核苷酸中均可显示，但表明添加到双链体的第一个nt与未连接寡核苷酸的3'nt之间需要互补。这些结果表明，对于PolIk的3'-核酸外切酶缺陷形式进行体外DNA合成，模板链磷酸二酯主链的连续性并非绝对必要。
