Elongation of palindromic repetitive DNA by DNA polymerase from hyperthermophilic archaea: A mechanism of DNA elongation and diversification

Abstract

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 °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′GA TTC3′) 6, the product was (5′GATATC3′) n , which had a different motif sequence from that of the seed oligoDNA. When a pseudopalindrome (5′AGATATC A3′) 6 was added to the reaction, the products were 5′TATC A · (AGATATC A) 3 · AGATATCT · ( TGATATCT) 5 · TGATA3′, etc. When 5′AGATATC A · ( AGATATCT3′) 5 was added, products were 5′TATCT · ( AGATATCT) 2 · TGATATCT · AGATATCT · AGATATC A · 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.