Ogata Norio
Research Institute, Taiko Pharmaceutical Co., Ltd., 3-34-14 Uchihonmachi, Suita, Osaka 564-0032, Japan.
Biochimie. 2007 May;89(5):702-12. doi: 10.1016/j.biochi.2006.12.011. Epub 2007 Jan 13.
DNA polymerase from hyperthermophilic bacteria can elongate tandem repetitive oligoDNA with a complete or incomplete palindromic sequence under isothermal conditions by "hairpin elongation". However, the product of the reaction has not yet been sufficiently characterized. Here, I demonstrate that when palindromic repetitive oligoDNA, e.g., (5'AGATATCT3')(6), was added as a "seed" to the DNA synthesis reaction catalyzed by DNA polymerase from the archaea Thermococcus litoralis (Vent polymerase) at 74 degrees C, the product was (5'AGATATCT3')(n). The product itself was palindromic and repetitive, and its motif (unit) sequence was exactly the same as that of the seed oligoDNA. On the other hand, when a pseudopalindrome, which contains a palindrome-breaking nucleotide (underlined), was present in seed oligoDNA, e.g., (5'GATTC3')(6), the product was (5'GATATC3')(n), which had a different motif sequence from that of the seed oligoDNA. When a pseudopalindrome (5'AGATATCA3')(6) was added to the reaction, the products were 5'TATCA . (AGATATCA)(3) . AGATATCT . (TGATATCT)(5) . TGATA3', etc. When 5'AGATATCA . (AGATATCT3')(5) was added, products were 5'TATCT . (AGATATCT)(2).TGATATCT . AGATATCT . AGATATCA . AGATATCT . AGA3', etc., demonstrating the generation of many "mutations" in the product DNA. I conclude that a tandem repetitive sequence is faithfully elongated (amplified) by hyperthermophilic DNA polymerase if it is completely palindromic, but is elongated with many errors if it is incompletely palindromic (pseudopalindromic) or mixed with a pseudopalindrome. The results suggest a protein-catalyzed elongation/diversification mechanism of short repetitive DNAs on the early earth.
嗜热菌的DNA聚合酶能够在等温条件下通过“发夹延伸”使具有完整或不完整回文序列的串联重复寡聚DNA得以延伸。然而，该反应的产物尚未得到充分表征。在此，我证明，当将回文重复寡聚DNA，例如(5'AGATATCT3')(6)作为“种子”添加到由嗜热栖热放线菌（Vent聚合酶）的DNA聚合酶在74摄氏度催化的DNA合成反应中时，产物为(5'AGATATCT3')(n)。产物本身是回文且重复的，其基序（单元）序列与种子寡聚DNA的完全相同。另一方面，当种子寡聚DNA中存在包含破坏回文核苷酸（下划线）的假回文时，例如(5'GATTC3')(6)，产物为(5'GATATC3')(n)，其基序序列与种子寡聚DNA的不同。当将假回文(5'AGATATCA3')(6)添加到反应中时，产物为5'TATCA.(AGATATCA)(3).AGATATCT.(TGATATCT)(5).TGATA3'等。当添加5'AGATATCA.(AGATATCT3')(5)时，产物为5'TATCT.(AGATATCT)(2).TGATATCT.AGATATCT.AGATATCA.AGATATCT.AGA3'等，这表明产物DNA中产生了许多“突变”。我得出结论，如果串联重复序列是完全回文的，则嗜热DNA聚合酶会忠实地使其延伸（扩增），但如果它是不完全回文（假回文）或与假回文混合，则会有许多错误地延伸。这些结果提示了早期地球上短重复DNA的蛋白质催化延伸/多样化机制。
