Cooperative DnaA Binding to the Negatively Supercoiled datA Locus Stimulates DnaA-ATP Hydrolysis

Abstract

Edited by Xiao-Fan WangTimely initiation of replication in Escherichia coli requires functional regulation of the replication initiator, ATP-DnaA. The cellular level of ATP-DnaA increases just before initiation, after which its level decreases through hydrolysis of DnaAbound ATP, yielding initiation-inactive ADP-DnaA. Previously, we reported a novel DnaA-ATP hydrolysis system involving the chromosomal locus datA and named it datA-dependent DnaA-ATP hydrolysis (DDAH). The datA locus contains a binding site for a nucleoid-associating factor integration host factor (IHF) and a cluster of three known DnaA-binding sites, which are important for DDAH. However, the mechanisms underlying the formation and regulation of the datA-IHF⅐DnaA complex remain unclear. We now demonstrate that a novel DnaA box within datA is essential for ATP-DnaA complex formation and DnaA-ATP hydrolysis. Specific DnaA residues, which are important for interaction with bound ATP and for head-to-tail inter-DnaA interaction, were also required for ATP-DnaA-specific oligomer formation on datA. Furthermore, we show that negative DNA supercoiling of datA stabilizes ATP-DnaA oligomers, and stimulates datA-IHF interaction and DnaA-ATP hydrolysis. Relaxation of DNA supercoiling by the addition of novobiocin, a DNA gyrase inhibitor, inhibits datA function in cells. On the basis of these results, we propose a mechanistic model of datA-IHF⅐DnaA complex formation and DNA supercoiling-dependent regulation for DDAH.In Escherichia coli, the ATP-bound DnaA protein (ATPDnaA) 3 has an essential role in assembly of initiation complexes on the chromosomal replication origin oriC (1-5). oriC consists of an AT-rich duplex-unwinding element (DUE) and a DnaA oligomerization region that contains two subregions with oppositely oriented clusters of 9-bp DnaA-binding sites (DnaA boxes) (see Fig. 1A). DnaA boxes R1 and R4 at the outer edges of the DnaA oligomerization region are oriented oppositely and have the highest affinities for DnaA of the sequences in this region. Cooperative binding of ATP-DnaA molecules to lower affinity sites in each subregion of oriC results in formation of left-half and right-half DnaA subcomplexes (6 -8). The lefthalf subcomplex binds to the region of oriC that contains the DUE and executes unwinding of DNA within the element (6, 9). IHF, a nucleoid-associated protein (NAP), binds to a specific IHF-binding site (IBS; see Fig. 1A) within the left-half subregion of oriC and bends DNA sharply (ϳ180°), enhancing ATP-DnaA binding and duplex unwinding (9, 10). The DnaA subcomplexes also interact with a pair of DnaB helicases for loading of those to the single-stranded DNA (9,11,12).DnaA consists of four domains (3). The N-terminal domain I has a DnaB-binding site (12). Domain II is a flexible linker (12). Domain III is the AAAϩ domain that has motifs for binding and hydrolysis of ATP (Walker A and B and Sensor I and II) and for interaction between DnaA protomers (arginine-finger, Box VII, AID-1, and AID-2) (see Fig. 1B) (6, 13-16). DnaA argininefin...

Emerging roles for R-loop structures in the management of topological stress

“…Nucleosomes, for instance, preferentially form on negatively supercoiled DNA, with each nucleosomebinding event stabilizing one negative superhelical turn (29,30). The replication initiation proteins of E. coli (DnaA) and of Drosophila (ORC1), along with a number of transcription factors (31)(32)(33)(34)(35)(36), also prefer binding to negatively supercoiled DNA. Negatively supercoiled topological domains therefore represent hubs of genome organization and activity in both prokaryotes and eukaryotes (37,38).…”

Coordination betweenE. coliCell Size and Cell Cycle Mediated by DnaA

“…At faster cell growth, the dosage of datA is higher, then the titration role is stronger, and finally, the effect on DnaA dynamics is higher when datA is moved closer to the terminus. It was also reported that datA can hydrolyze DnaA-ATP independent of Hda-β-clamp-mediated RIDA, but datA-mediated hydrolysis is weaker than Hda-β-clamp-mediated RIDA [91,92]. Considering this fact in the model may make the predictions of datA perturbations more comparable to experimental data.…”

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