State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Chaoyang District, Beijing 100101, China.
Nucleic Acids Res. 2012 Apr;40(8):3470-83. doi: 10.1093/nar/gkr1256. Epub 2011 Dec 20.
The eukaryotic-like primase from the hyperthermophilic archaeon Sulfolobus solfataricus (SsoPriSL) exhibits a range of activities including template-dependent de novo primer synthesis, primer extension and template-independent terminal nucleotidyl transfer using either rNTPs or dNTPs. Remarkably, the enzyme is able to synthesize products far longer than templates in vitro. Here we show that the long products resulted from template-dependent polymerization across discontinuous templates (PADT) by SsoPriSL. PADT was initiated through either primer synthesis or terminal transfer, and occurred efficiently on templates containing contiguous dCs. Template switching took place when the 3'-end of a growing strand synthesized on one template annealed to another template directly or following the terminal addition of nucleotides, and was subsequently extended on the new template. The key to PADT was the ability of SsoPriSL to promote strand annealing. SsoPriSL catalyzed PADT with either dNTPs or rNTPs as the substrates but preferred the latter. The enzyme remained active in PADT but became inefficient in primer synthesis in vitro when temperature was raised from 55°C to 70°C. Our results suggest that SsoPriSL is capable of bridging noncomplementary DNA ends and, therefore, may serve a role in double-strand DNA break repair in Archaea.
嗜热古菌 Sulfolobus solfataricus 的真核样引物酶（SsoPriSL）表现出多种活性，包括模板依赖性从头引物合成、引物延伸以及使用 rNTP 或 dNTP 进行模板非依赖性末端核苷酸转移。值得注意的是，该酶能够在体外合成远远长于模板的产物。在这里，我们证明了 SsoPriSL 通过模板依赖性聚合跨越不连续模板（PADT）产生长产物。PADT 通过引物合成或末端转移启动，在含有连续 dCs 的模板上高效发生。当一条在一个模板上合成的延伸链的 3'端退火到另一个模板上，或者在末端添加核苷酸后退火到另一个模板上，并且随后在新模板上延伸时，模板转换就会发生。PADT 的关键是 SsoPriSL 促进链退火的能力。SsoPriSL 可以催化 dNTP 或 rNTP 作为底物进行 PADT，但更喜欢后者。当温度从 55°C 升高到 70°C 时，该酶在 PADT 中保持活性，但在体外的引物合成中效率降低。我们的结果表明，SsoPriSL 能够桥接非互补 DNA 末端，因此可能在古菌的双链 DNA 断裂修复中发挥作用。
