Near-Atomic Structural Model for Bacterial DNA Replication Initiation Complex and its Functional Insights

Abstract

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.DnaA | molecular simulation | coarse-grained model | oriC C hromosomal DNA replication is initiated by unwinding the dsDNA of the replication origin, which requires formation of higher-order protein-DNA complexes, typically referred to as the initiation complexes (1). DnaA is a major replication initiation protein conserved in the initiation complex of most eubacterial species. In a model organism, Escherichia coli, DnaA forms homooligomers on the replication origin oriC, which promotes dsDNA unwinding. The resulting ssDNA is captured by DnaB helicase, followed by formation of the replisomes (2-5). Molecular mechanisms of how DnaA facilitates dsDNA unwinding are still unclear, although some models have been proposed (1, 6-10). A high-resolution structure model of the initiation complex, discovered using computational modeling based on experimental data, would provide significant insight into the molecular mechanism.The E. coli minimal oriC region contains the AT-rich DNA unwinding element (DUE), at least 11 DnaA-binding motifs (termed DnaA boxes) and a single binding site for the integration host factor (IHF) (Fig. 1A) (1-5, 11-15). The DnaA boxes contain 9-mer nucleotides (consensus sequence TTATNCACA, where N can be any base) (16). The 11 DnaA boxes (R1-2, R4, R5M, I1-3, C1-3, and τ2) have differing affinities to DnaA and motif orientations (indicated by triangles in Fig. 1A): The two terminal boxes R1 and R4 have especially high affinities (dissociation constants 1-6 nM for R1 and ∼1 nM for R4), whereas others have modest (R2) to low affinities (I1-3, C1-3, and R5M and τ2; dissociation constants for R5M are >200 nM) (12-19). The 11 DnaA boxes have been divided into two groups...

Identification of a basal system for unwinding a bacterial chromosome origin

“…The proximity of the specific DNA sequences within the B. subtilis unwinding region suggested a mechanism whereby DnaA proteins bound to DnaA‐box#6/7 guide the formation of a DnaA filament onto the adjacent DnaA‐trios. However, it has been proposed that during replication initiation in E. coli, DnaA binds DnaA‐boxes upstream of the unwinding site and engages ssDNA that is delivered by the bending protein IHF, thereby simultaneously contacting dsDNA and ssDNA (Ozaki & Katayama, ; Noguchi et al , ; Shimizu et al , ; Sakiyama et al , ). Although the minimal artificial unwinding sequence of B. subtilis used in vivo did not contain specific DnaA binding sites upstream of DnaA‐box#6/7, it remained possible that DnaA could bind upstream through either formation of a DnaA filament on dsDNA emanating from DnaA‐box#6 (Erzberger et al , ; Scholefield et al , ) or stabilization by a partner DNA binding protein (Chodavarapu et al , ).…”

“…Second, the ability of the DnaA ATP to specifically bind and stretch ssDNA (Speck & Messer, ; Duderstadt et al , ; Cheng et al , ; Richardson et al , ) suggests that a DnaA filament could promote origin unwinding by assembling onto one strand of dsDNA and destabilizing the double helix. Third, a combination of biochemical, genetic and modelling data using the Escherichia coli replication system suggests that DnaA binds dsDNA as a monomer proximal to the unwinding region and as an oligomer distal to the unwinding region (Ozaki & Katayama, ; Kaur et al , ; Noguchi et al , ; Shimizu et al , ; Sakiyama et al , ). Subsequent binding of an architectural protein between the proximal and distal sites introduces a sharp bend in the dsDNA that connects DnaA proteins from the two regions to promote unwinding and recruit ssDNA to the dsDNA‐bound DnaA oligomer.…”

Regulation of replication initiation: lessons from <i>Caulobacter crescentus</i>

“…2). The mechanisms underlying formation of the initiation complex have been characterized in Escherichia coli (Ozaki and Katayama, 2009;Kaguni, 2011;Leonard and Grimwade, 2015;Katayama et al, 2017); in this organism, the 245-bp minimal origin (oriC) contains 11 functional DnaA boxes as well as the binding site (IBS) for integration host factor (IHF), a nucleoid-associated protein that creates sharp kinks in DNA (McGarry et al, 2004;Kawakami et al, 2005;Rozgaja et al, 2011;Shimizu et al, 2016). DnaA boxes at the edges of the DnaA assembly regions have the highest affinity for DnaA with K d values of 3-9 nM (Ozaki and Katayama, 2009).…”

Molecular determinants of the Bacillus subtilis chromosome origin basal unwinding system

“…Based mainly on studies using Escherichia coli, in one model DnaA forms an oligomer on dsDNA that simultaneously binds ssDNA. Here a DnaA ATP oligomer assembles at DnaA-boxes upstream of the oriC unwinding site and then interacts with a single DNA strand from downstream that is delivered by a loop mediated by the architectural protein IHF (Noguchi et al, 2015;Ozaki and Katayama, 2012;Sakiyama et al, 2017;Shimizu et al, 2016). Alternatively, based mainly on studies of Aquifex aeolicus, an opposing model posits that DnaA binding to dsDNA and ssDNA is mutually exclusive.…”