Nitharwal Ram Gopal, Paul Subhankar, Dar Ashraf, Choudhury Nirupam Roy, Soni Rajesh K, Prusty Dhaneswar, Sinha Sukrat, Kashav Tara, Mukhopadhyay Gauranga, Chaudhuri Tapan Kumar, Gourinath Samudrala, Dhar Suman Kumar
Special Centre for Molecular Medicine, Jawaharlal Nehru University, New Delhi 110067, India.
Nucleic Acids Res. 2007;35(9):2861-74. doi: 10.1093/nar/gkm167. Epub 2007 Apr 11.
Hexameric DnaB type replicative helicases are essential for DNA strand unwinding along with the direction of replication fork movement. These helicases in general contain an amino terminal domain and a carboxy terminal domain separated by a linker region. Due to the lack of crystal structure of a full-length DnaB like helicase, the domain structure and function of these types of helicases are not clear. We have reported recently that Helicobacter pylori DnaB helicase is a replicative helicase in vitro and it can bypass Escherichia coli DnaC activity in vivo. Using biochemical, biophysical and genetic complementation assays, here we show that though the N-terminal region of HpDnaB is required for conformational changes between C6 and C3 rotational symmetry, it is not essential for in vitro helicase activity and in vivo function of the protein. Instead, an extreme carboxy terminal region and an adjacent unique 34 amino acid insertion region were found to be essential for HpDnaB activity suggesting that these regions are important for proper folding and oligomerization of this protein. These results confer great potential in understanding the domain structures of DnaB type helicases and their related function.
六聚体DnaB型复制解旋酶对于沿复制叉移动方向解开DNA链至关重要。这些解旋酶通常包含一个氨基末端结构域和一个羧基末端结构域，中间由一个连接区域隔开。由于缺乏全长类DnaB解旋酶的晶体结构，这些类型解旋酶的结构域结构和功能尚不清楚。我们最近报道，幽门螺杆菌DnaB解旋酶在体外是一种复制解旋酶，并且在体内能够绕过大肠杆菌DnaC的活性。通过生化、生物物理和遗传互补分析，我们在此表明，虽然HpDnaB的N端区域对于C6和C3旋转对称性之间的构象变化是必需的，但对于该蛋白质的体外解旋酶活性和体内功能并非必不可少。相反，发现一个极端的羧基末端区域和一个相邻的独特34个氨基酸插入区域对于HpDnaB活性至关重要，这表明这些区域对于该蛋白质的正确折叠和寡聚化很重要。这些结果为理解DnaB型解旋酶的结构域结构及其相关功能提供了巨大潜力。
