Islas L, Fairley C F, Morgan W F
Department of Radiation Oncology, University of California, San Francisco, CA 94143-0750, USA.
Nucleic Acids Res. 1998 Aug 15;26(16):3729-38. doi: 10.1093/nar/26.16.3729.
DNA polymerases catalyze the synthesis of DNA using a continuous uninterrupted template strand. However, it has been shown that a 3'-->5' exonuclease-deficient form of the Klenow fragment of Escherichia coli DNA polymerase I as well as DNA polymerase of Thermus aquaticus can synthesize DNA across two unlinked DNA templates. In this study, we used an oligonucleotide-based assay to show that discontinuous DNA synthesis was present in HeLa cell extracts. DNA synthesis inhibitor studies as well as fractionation of the extracts revealed that most of the discontinuous DNA synthesis was attributable to DNA polymerase alpha. Additionally, discontinuous DNA synthesis could be eliminated by incubation with an antibody that specifically neutralized DNA polymerase alpha activity. To test the relative efficiency of each nuclear DNA polymerase for discontinuous synthesis, equal amounts (as measured by DNA polymerase activity) of DNA polymerases alpha, beta, delta (+/- PCNA) and straightepsilon (+/- PCNA) were used in the discontinuous DNA synthesis assay. DNA polymerase alpha showed the most discontinuous DNA synthesis activity, although small but detectable levels were seen for DNA polymerases delta (+PCNA) and straightepsilon (- PCNA). Klenow fragment and DNA polymerase beta showed no discontinuous DNA synthesis, although at much higher amounts of each enzyme, discontinuous synthesis was seen for both. Discontinuous DNA synthesis by DNA polymerase alpha was seen with substrates containing 3 and 4 bp single-strand stretches of complementarity; however, little synthesis was seen with blunt substrates or with 1 bp stretches. The products formed from these experiments are structurally similar to that seen in vivo for non-homologous end joining in eukaryotic cells. These data suggest that DNA polymerase alpha may be able to rejoin double-strand breaks in vivo during replication.
DNA聚合酶利用连续不间断的模板链催化DNA的合成。然而，研究表明，大肠杆菌DNA聚合酶I的Klenow片段的3'→5'核酸外切酶缺陷型以及嗜热水生菌的DNA聚合酶能够跨越两个不相连的DNA模板合成DNA。在本研究中，我们使用基于寡核苷酸的检测方法表明，HeLa细胞提取物中存在不连续的DNA合成。DNA合成抑制剂研究以及提取物的分级分离表明，大部分不连续的DNA合成归因于DNA聚合酶α。此外，通过与特异性中和DNA聚合酶α活性的抗体孵育，可以消除不连续的DNA合成。为了测试每种核DNA聚合酶进行不连续合成的相对效率，在不连续DNA合成检测中使用了等量（通过DNA聚合酶活性测量）的DNA聚合酶α、β、δ（±增殖细胞核抗原）和ε（±增殖细胞核抗原）。DNA聚合酶α显示出最高的不连续DNA合成活性，尽管DNA聚合酶δ（+增殖细胞核抗原）和ε（-增殖细胞核抗原）也有少量但可检测到的活性。Klenow片段和DNA聚合酶β没有显示出不连续的DNA合成，尽管在每种酶的量高得多时，两者都出现了不连续合成。DNA聚合酶α在含有3和4个碱基对互补单链延伸的底物上表现出不连续DNA合成；然而，在平端底物或1个碱基对延伸的底物上几乎没有合成。这些实验形成的产物在结构上与真核细胞中非同源末端连接在体内所见的产物相似。这些数据表明，DNA聚合酶α可能能够在体内复制过程中重新连接双链断裂。
