Enhancement of DNA, cDNA synthesis and fidelity at high temperatures by a dimeric single-stranded DNA-binding protein
Enhancement of DNA, cDNA synthesis and fidelity at high temperatures by a dimeric single-stranded DNA-binding protein
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DOI：
10.1093/nar/gkg865
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发表时间：
2003-11-15
影响因子：
14.9
通讯作者：
Berenguer, J
中科院分区：
文献类型：
--
作者：
Perales, C;Cava, F;Berenguer, J
细菌单链DNA结合蛋白（SSBs）是DNA复制和修复所必需的。我们从嗜热热菌（Thermus thermophilus, TthSSB）中过表达和纯化了SSB的天然形式和两个his标记的融合体。在溶液中发现这三种蛋白为二聚体。它们在体外与单链DNA特异性结合，温度范围为4-80度，野生型蛋白可以承受94度的孵育2分钟。在PCR中加入TthSSB使嗜热t球菌（Tth）和炽热焦球菌（Pfu）的DNA聚合酶合成PCR中的DNA片段所需的延伸时间缩短了一半。TthSSB的存在增加了嗜热单胞菌无校对DNA聚合酶的保真度。TthSSB还能够结合单链RNA，从而在cDNA合成过程中显著增强其同源Tth DNA聚合酶的逆转录活性。
Bacterial single-stranded DNA-binding proteins (SSBs) are required for DNA replication and repair. We have over-expressed and purified the native form and two His-tagged fusions of the SSB from Thermus thermophilus (TthSSB). The three proteins were found as dimers in solution. They bound in vitro to single-stranded DNA specifically over a temperature range of 4-80degreesC, and the wild-type protein could withstand incubation at 94degreesC for 2 min. Addition of TthSSB to PCR halved the elongation time required for the DNA polymerases of T.thermophilus (Tth) and Pyrococcus furiosus (Pfu) to synthesise DNA fragments in PCRs. The presence of TthSSB increased the fidelity of the proof- reading-free DNA polymerase of T.thermophilus. TthSSB was also able to bind single-stranded RNA, allowing a dramatic enhancement of the reverse transcription activity of its cognate Tth DNA polymerase during cDNA synthesis.