Mutant DnaAs of Escherichia coli that are refractory to negative control

Abstract

DnaA is the initiator of DNA replication in bacteria. A mutant DnaA named DnaAcos is unusual because it is refractory to negative regulation. We developed a genetic method to isolate other mutant DnaAs that circumvent regulation to extend our understanding of mechanisms that control replication initiation. Like DnaAcos, one mutant bearing a tyrosine substitution for histidine 202 (H202Y) withstands the regulation exerted by datA, hda and dnaN (β clamp), and both DnaAcos and H202Y resist inhibition by the Hda-β clamp complex in vitro. Other mutant DnaAs carrying G79D, E244K, V303M or E445K substitutions are either only partially sensitive or refractory to inhibition by the Hda-β clamp complex in vitro but are responsive to hda expression in vivo. All mutant DnaAs remain able to interact directly with Hda. Of interest, both DnaAcos and DnaAE244K bind more avidly to Hda. These mutants, by sequestrating Hda, may limit its availability to regulate other DnaA molecules, which remain active to induce extra rounds of DNA replication. Other evidence suggests that a mutant bearing a V292M substitution hyperinitiates by escaping the effect of an unknown regulatory factor. Together, our results provide new insight into the mechanisms that regulate replication initiation in Escherichia coli.

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

“…The oriC region was previously isolated as a suppressor of a dnaA cos mutant, which induced overinitiation of chromosomal replication and inhibited colony formation at 30°C ( Katayama and Kornberg, 1994 ). DnaAcos protein is resistant to RIDA and sustains the replication initiation activity at 30°C, which leads to overinitiation of replication ( Katayama, 1994 ; Katayama and Kornberg, 1994 ; Chodavarapu et al, 2013 ). It is suggested that the plasmid-borne multiple oriC copies titrate DnaA molecules, resulting in a decreased amount of DnaA available for chromosomal oriC binding and, therefore, inhibition of additional chromosomal replication initiations.…”

“…To explore the mechanism of hda-185 suppression by yfdQRST , we examined the effect of pSSPB and pSSPT in dnaAcos mutant cells. As described above, the initiation activity of DnaAcos is resistant to RIDA and is sustained over long periods at 30°C, resulting in overinitiation of replication and inhibition of colony formation ( Katayama and Kornberg, 1994 ; Katayama, 1994 ; Chodavarapu et al, 2013 ). If the mechanism of yfdQRST initiation inhibition was independent of RIDA, the presence of pSSPB and pSSPT would suppress the defects of dnaAcos cells.…”

The DnaA Tale

“…The first indication that each of the domains is essential for the activity of the DnaA protein came from the mapping and sequencing of the classical dnaA Ts mutants (Hansen et al, 1984 , 1992 ), see Figure 5 for position of the mutations and their amino acid changes. Since then many other dnaA mutants have been isolated both by different selections and by screening of in vitro generated mutants (e.g., reviewed in Erzberger et al, 2002 ; and additional mutants described in the following citations: Felczak and Kaguni, 2004 ; Asklund and Atlung, 2005 ; Felczak et al, 2005 ; Chodavarapu et al, 2013 ).…”

Caulobacter crescentus β sliding clamp employs a noncanonical regulatory model of DNA replication

“…Since the binding pocket is required for the polymerase tethering, clamp loading, and HdaA association, both DNA replication and RIDA were predicted to be less active when Cc DnaN contained a mutated novel pocket. To verify this, in vitro replication and RIDA assays were performed using previously developed methods .…”