Antipova Valeriya N, Zheleznaya Lyudmila A, Zyrina Nadezhda V
Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Pushchino, Moscow Region, Russia.
FEMS Microbiol Lett. 2014 Aug;357(2):144-50. doi: 10.1111/1574-6968.12511. Epub 2014 Jul 14.
In the absence of added DNA, thermophilic DNA polymerases synthesize double-stranded DNA from free dNTPs, which consist of numerous repetitive units (ab initio DNA synthesis). The addition of thermophilic restriction endonuclease (REase), or nicking endonuclease (NEase), effectively stimulates ab initio DNA synthesis and determines the nucleotide sequence of reaction products. We have found that NEases Nt.AlwI, Nb.BbvCI, and Nb.BsmI with non-palindromic recognition sites stimulate the synthesis of sequences organized mainly as palindromes. Moreover, the nucleotide sequence of the palindromes appeared to be dependent on NEase recognition/cleavage modes. Thus, the heterodimeric Nb.BbvCI stimulated the synthesis of palindromes composed of two recognition sites of this NEase, which were separated by AT-reach sequences or (A)n (T)m spacers. Palindromic DNA sequences obtained in the ab initio DNA synthesis with the monomeric NEases Nb.BsmI and Nt.AlwI contained, along with the sites of these NEases, randomly synthesized sequences consisted of blocks of short repeats. These findings could help investigation of the potential abilities of highly productive ab initio DNA synthesis for the creation of DNA molecules with desirable sequence.
在没有添加DNA的情况下，嗜热DNA聚合酶可从游离的dNTP合成双链DNA，这些dNTP由众多重复单元组成（从头DNA合成）。添加嗜热限制性内切酶（REase）或切口内切酶（NEase）可有效刺激从头DNA合成，并确定反应产物的核苷酸序列。我们发现，具有非回文识别位点的NEases Nt.AlwI、Nb.BbvCI和Nb.BsmI可刺激主要组织为回文的序列的合成。此外，回文的核苷酸序列似乎取决于NEase的识别/切割模式。因此，异二聚体Nb.BbvCI刺激了由该NEase的两个识别位点组成回文的合成，这两个识别位点被富含AT的序列或（A）n（T）m间隔隔开。在与单体NEases Nb.BsmI和Nt.AlwI进行的从头DNA合成中获得的回文DNA序列，除了这些NEase的位点外，还包含由短重复序列块组成的随机合成序列。这些发现有助于研究高效从头DNA合成在创建具有理想序列的DNA分子方面的潜在能力。
