Toth Eric A, Li Ying, Sawaya Michael R, Cheng Yifan, Ellenberger Tom
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA.
Mol Cell. 2003 Nov;12(5):1113-23. doi: 10.1016/s1097-2765(03)00442-8.
Within minutes after infecting Escherichia coli, bacteriophage T7 synthesizes many copies of its genomic DNA. The lynchpin of the T7 replication system is a bifunctional primase-helicase that unwinds duplex DNA at the replication fork while initiating the synthesis of Okazaki fragments on the lagging strand. We have determined a 3.45 A crystal structure of the T7 primase-helicase that shows an articulated arrangement of the primase and helicase sites. The crystallized primase-helicase is a heptamer with a crown-like shape, reflecting an intimate packing of helicase domains into a ring that is topped with loosely arrayed primase domains. This heptameric isoform can accommodate double-stranded DNA in its central channel, which nicely explains its recently described DNA remodeling activity. The double-jointed structure of the primase-helicase permits a free range of motion for the primase and helicase domains that suggests how the continuous unwinding of DNA at the replication fork can be periodically coupled to Okazaki fragment synthesis.
在感染大肠杆菌后的几分钟内，噬菌体T7会合成许多拷贝的基因组DNA。T7复制系统的关键是一种双功能引发酶-解旋酶，它在复制叉处解开双链DNA，同时在滞后链上启动冈崎片段的合成。我们确定了T7引发酶-解旋酶的3.45埃晶体结构，该结构显示了引发酶和解旋酶位点的铰接排列。结晶的引发酶-解旋酶是一种七聚体，呈冠状，反映了解旋酶结构域紧密堆积成环，环顶部排列着松散的引发酶结构域。这种七聚体异构体可以在其中心通道容纳双链DNA，这很好地解释了其最近描述的DNA重塑活性。引发酶-解旋酶的双关节结构允许引发酶和解旋酶结构域自由运动，这表明了复制叉处DNA的连续解旋如何能够周期性地与冈崎片段合成相耦合。
