SeqA Blocking of DnaA-oriC Interactions Ensures Staged Assembly of the E. coli Pre-RC

Abstract

DnaA occupies only the three highest-affinity binding sites in E. coli oriC throughout most of the cell cycle. Immediately prior to initiation of chromosome replication, DnaA interacts with additional recognition sites, resulting in localized DNA-strand separation. These two DnaA-oriC complexes formed during the cell cycle are functionally and temporally analogous to yeast ORC and pre-RC. After initiation, SeqA binds to hemimethylated oriC, sequestering oriC while levels of active DnaA are reduced, preventing reinitiation. In this paper, we investigate how resetting of oriC to the ORC-like complex is coordinated with SeqA-mediated sequestration. We report that oriC resets to ORC during sequestration. This was possible because SeqA blocked DnaA binding to hemimethylated oriC only at low-affinity recognition sites associated with GATC but did not interfere with occupation of higher-affinity sites. Thus, during the sequestration period, SeqA repressed pre-RC assembly while ensuring resetting of E. coli ORC.

A Role for Nonessential Domain II of Initiator Protein, DnaA, in Replication Control

“…The SeqA protein sequesters the origin immediately after initiation due to its cooperative binding to hemi-methylated GATC sites, found in abundance near the origin (Campbell and Kleckner 1990;Lu et al 1994;von Freiesleben et al 2000). During this period, DnaA is bound only to its high-affinity sites in oriC (sites that bind DnaA in both ATP and ADP forms) and is precluded from binding to lower affinity sites (bound only by ATP-DnaA), whose occupancy is necessary for origin firing (Nievera et al 2006). In seqA mutants, DnaA binds prematurely to lowaffinity sites (Nievera et al 2006), with the resulting asynchronous and premature initiation of replication.…”

“…During this period, DnaA is bound only to its high-affinity sites in oriC (sites that bind DnaA in both ATP and ADP forms) and is precluded from binding to lower affinity sites (bound only by ATP-DnaA), whose occupancy is necessary for origin firing (Nievera et al 2006). In seqA mutants, DnaA binds prematurely to lowaffinity sites (Nievera et al 2006), with the resulting asynchronous and premature initiation of replication.…”

Initiating chromosome replication in E. coli: it makes sense to recycle

“…During this period, SeqA blocks dnaA transcription (Campbell and Kleckner 1990) as well as DnaA-ATP rebinding to the low-affinity sites in oriC (Fig. 1, stage 3; Nievera et al 2006). GATC is not found at R1, R2, and R4 in oriC, allowing immediate reset of ORC during the sequestration period (Fig.…”

“…An early step toward accomplishing this task requires assembly of initiator protein complexes that prepare replication origins to begin new rounds of DNA replication (Bell 2002;Leonard and Grimwade 2005;Mott and Berger 2007). Each origin is initially marked by a multisubunit origin recognition complex (ORC) that recruits additional initiator proteins and transitions into a prereplication complex (pre-RC) responsible for loading the replicative helicase (Stillman 2005;Nievera et al 2006). It is now clear that initiator proteins from all domains of life share structural and functional similarities, including membership in the AAA + family of proteins whose activity is regulated by binding and hydrolysis of ATP (Erzberger et al 2002).…”

“…These DNA-protein complexes form a eubacterial version of ORC (Fig. 1, stage 1; Nievera et al 2006). The transition to a pre-RC requires recruitment of DnaA-ATP to low-affinity 9-mer recognition sites (I sites, t sites, R5M, and R3) within inter-ORC DNA (Fig.…”