Nucleotide-Induced Conformational Changes in Escherichia coli DnaA Protein Are Required for Bacterial ORC to Pre-RC Conversion at the Chromosomal Origin

Abstract

DnaA oligomerizes when bound to origins of chromosomal replication. Structural analysis of a truncated form of DnaA from Aquifex aeolicus has provided insight into crucial conformational differences within the AAA+ domain that are specific to the ATP- versus ADP- bound form of DnaA. In this study molecular docking of ATP and ADP onto Escherichia coli DnaA, modeled on the crystal structure of Aquifex aeolicus DnaA, reveals changes in the orientation of amino acid residues within or near the vicinity of the nucleotide-binding pocket. Upon limited proteolysis with trypsin or chymotrypsin ADP-DnaA, but not ATP-DnaA generated relatively stable proteolytic fragments of various sizes. Examined sites of limited protease susceptibility that differ between ATP-DnaA and ADP-DnaA largely reside in the amino terminal half of DnaA. The concentration of adenine nucleotide needed to induce conformational changes, as detected by these protease susceptibilities of DnaA, coincides with the conversion of an inactive bacterial origin recognition complex (bORC) to a replication efficient pre-replication complex (pre-RC) at the E. coli chromosomal origin of replication (oriC).

Origin recognition is the predominant role for DnaA-ATP in initiation of chromosome replication

“…Consistent with this idea, the A in position 7 in the consensus sequence 5′- TTA/TTNCACA, which aids sequence-specific binding to domain IV ( 47 ), is a common feature among 9 mer sequences that bind DnaA-ADP. It is also possible that some conformational changes in DnaA, observed at physiological ATP concentrations (5 mM) ( 48 ), contribute to the cooperative binding that extends DnaA between low affinity DnaA-ATP sites. It should also be noted that DnaA-ATP sites have not been identified in many bacterial origins, and some bacterial DnaAs do not contain a domain 1 that is capable of DnaA–DnaA interactions ( 49 ).…”

The Escherichia coli Cryptic Prophage Protein YfdR Binds to DnaA and Initiation of Chromosomal Replication Is Inhibited by Overexpression of the Gene Cluster yfdQ-yfdR-yfdS-yfdT

“…In Escherichia coli , the initiator protein DnaA recognizes the chromosomal replication origin, oriC ( Ozaki and Katayama, 2009 ; Kaguni, 2011 ; Leonard and Grimwade, 2011 ; Costa et al, 2013 ; Saxena et al, 2013 ). DnaA has a high affinity for both ATP and ADP ( Sekimizu et al, 1987 ; Saxena et al, 2015 ). ATP-bound DnaA (ATP-DnaA), but not ADP-bound DnaA (ADP-DnaA), forms stable multimers on oriC and induces unwinding of a specific AT-rich oriC region, depending on the oriC binding of the nucleotide-associating protein IHF ( Ryan et al, 2002 ; Dillon and Dorman, 2010 ; Ozaki and Katayama, 2012 ).…”

Replisome Assembly at Bacterial Chromosomes and Iteron Plasmids

“…ATP- Ec DnaA form of initiator binds efficiently both high and low affinity binding sites (McGarry et al, 2004 ; Kawakami et al, 2005 ). Based on molecular docking, binding of ATP, instead of ADP, is presumed to cause changes in the Ec DnaA protein conformation, thus leading to the formation of large oligomeric complex within the origin region (Saxena et al, 2015 ). The crystallographic data, when nonhydrolyzable ATP analog AMP-PCP was used, showed the formation of open-ended, right-handed helical filament of Aa DnaA (Erzberger et al, 2006 ).…”