期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2016
卷号：113
期号：50
页码：E8021-E8030
DOI：10.1073/pnas.1609649113
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：SignificanceChromosomal DNA replication is initiated by unwinding duplex DNA via formation of dynamic higher-order protein-DNA complexes. Whereas biochemical analyzes have been performed for a long time, structural dynamics in those processes still remain elusive due to their complex nature. Here, using hybrid approaches of computational modeling and biochemical assays, we explore dynamic structures of the entire machinery and significance of spatial arrangement of every component in the replication initiation complex of Escherichia coli. We show the structural model of the crucial core part of the complex at near-atomic resolution. The complex is composed of three subcomplexes, and spatial arrangements of those as well as their components are important for efficient replication initiation. Upon DNA replication initiation in Escherichia coli, the initiator protein DnaA forms higher-order complexes with the chromosomal origin oriC and a DNA-bending protein IHF. Although tertiary structures of DnaA and IHF have previously been elucidated, dynamic structures of oriC-DnaA-IHF complexes remain unknown. Here, combining computer simulations with biochemical assays, we obtained models at almost-atomic resolution for the central part of the oriC-DnaA-IHF complex. This complex can be divided into three subcomplexes; the left and right subcomplexes include pentameric DnaA bound in a head-to-tail manner and the middle subcomplex contains only a single DnaA. In the left and right subcomplexes, DnaA ATPases associated with various cellular activities (AAA+) domain III formed helices with specific structural differences in interdomain orientations, provoking a bend in the bound DNA. In the left subcomplex a continuous DnaA chain exists, including insertion of IHF into the DNA looping, consistent with the DNA unwinding function of the complex. The intervening spaces in those subcomplexes are crucial for DNA unwinding and loading of DnaB helicases. Taken together, this model provides a reasonable near-atomic level structural solution of the initiation complex, including the dynamic conformations and spatial arrangements of DnaA subcomplexes.
关键词：DnaA ; molecular simulation ; coarse-grained model ; oriC
