School of Biological Sciences, University of the Punjab, Lahore, 54590, Pakistan.
Folia Microbiol (Praha). 2020 Apr;65(2):407-415. doi: 10.1007/s12223-019-00745-9. Epub 2019 Aug 10.
Flap endonuclease is a structure-specific nuclease which cleaves 5'-flap of bifurcated DNA substrates. Genome sequence of Thermococcus kodakarensis harbors an open reading frame, Tk1281, exhibiting high homology with archaeal flap endonucleases 1. The corresponding gene was cloned and expressed in Escherichia coli, and the gene product was purified to apparent homogeneity. Tk1281 was a monomer of 38 kDa and catalyzed the cleavage of 5'-flap from double-stranded DNA substrate containing single-stranded DNA flap. The highest cleavage activity was observed at 80 °C and pH 7.5. Under optimal conditions, Tk1281 exhibited apparent V and K values of 278 nmol/min/mg and 37 μM, respectively, against a 54-nucleotide double-stranded substrate containing a single-stranded 5'-flap of 27 nucleotides. A unique feature of Tk1281 is its highest activation in the presence of Co and no activation with Mn. To the best of our knowledge, this is the first cloning and characterization of a flap endonuclease from the genus Thermococcus.
解旋酶是一种结构特异性核酸内切酶，可切割分叉 DNA 底物的 5'-发夹结构。Thermococcus kodakarensis 的基因组序列包含一个开放阅读框 Tk1281，与古菌解旋酶 1 具有高度同源性。该基因在大肠杆菌中被克隆和表达，并被纯化至明显的均一性。Tk1281 是一个 38 kDa 的单体，可催化双链 DNA 底物中单链 DNA 发夹的 5'-切割。在 80°C 和 pH 7.5 下观察到最高的切割活性。在最佳条件下，Tk1281 对含有 27 个核苷酸单链 5'-发夹的 54 个核苷酸双链底物的表观 V 和 K 值分别为 278 nmol/min/mg 和 37 μM。Tk1281 的一个独特特征是在 Co 存在下的最高激活，而在 Mn 存在下没有激活。据我们所知，这是首次从 Thermococcus 属中克隆和表征解旋酶。
