Membrane-mediated Release of Nucleotide from an Initiator of Chromosomal Replication, Escherichia coli DnaA, Occurs with Insertion of a Distinct Region of the Protein into the Lipid Bilayer

Garner, Jennifer; Durrer, Peter; Kitchen, Jennifer L.; Brunner, Josef; Crooke, Elliott

doi:10.1074/jbc.273.9.5167

Cited by 60 publications

(67 citation statements)

References 39 publications

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“…In E. coli, DnaA is thought to bind directly to membranes (18,54), although membrane association of oriC is mediated primarily via SeqA and SeqB (60). In contrast, membrane association in B. subtilis is dependent on DnaB (23,66,74), and there is no evidence for a direct association between DnaA and the membrane.…”

Section: Membrane Attachmentmentioning

confidence: 81%

Chromosomal Replication Initiation Machinery of Low-G+C-Content Firmicutes

2012

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bMuch of our knowledge of the initiation of DNA replication comes from studies in the Gram-negative model organism Escherichia coli. However, the location and structure of the origin of replication within the E. coli genome and the identification and study of the proteins which constitute the E. coli initiation complex suggest that it might not be as universal as once thought. The archetypal low-G؉C-content Gram-positive Firmicutes initiate DNA replication via a unique primosomal machinery, quite distinct from that seen in E. coli, and an examination of oriC in the Firmicutes species Bacillus subtilis indicates that it might provide a better model for the ancestral bacterial origin of replication. Therefore, the study of replication initiation in organisms other than E. coli, such as B. subtilis, will greatly advance our knowledge and understanding of these processes as a whole. In this minireview, we highlight the structure-function relationships of the Firmicutes primosomal proteins, discuss the significance of their oriC architecture, and present a model for replication initiation at oriC. The Firmicutes are Gram-positive bacteria encompassing three major classes, Bacilli, Clostridia, and Mollicutes. They have a relatively low GϩC content in their genomes and are morphologically and physiologically diverse. Rod-shaped bacilli, spherical cocci, and aerobic, anaerobic spore-forming, and non-sporeforming bacteria are found in this group. They likely represent the most ancestral phylum of prokaryotes, with high-GϩC-content Gram-positive and Gram-negative bacteria having diverged from the Firmicutes at a later stage in evolution (13,70). Bacillus subtilis is the best studied of the Firmicutes and is widely considered the Gram-positive model bacterium, but other bacilli, streptococci, staphylococci, and clostridia have been extensively studied because of their medical and industrial importance.In addition to the universally conserved replication initiation protein DnaA (32, 65) and the replication restart protein PriA (17, 39), the low-GϩC-content Firmicutes generally have two unique essential genes, dnaD and dnaB, coding for the replication initiation proteins DnaD and DnaB, respectively ( Table 1) (note that DnaB in B. subtilis is unrelated to DnaB in Escherichia coli). In some cases-for example, in several Mollicutes-there are no distinct dnaD and dnaB genes, but there is, instead, a single gene annotated as dnaD-like which may combine both functions. No homologous proteins are found outside the Firmicutes, suggesting a replication initiation machinery distinctly different from those of other bacteria (31). In addition, there are a number of regulatory proteins which are not found in the Gram-negative model organism Escherichia coli, including YabA, Soj, SirA, and Spo0A, while other regulatory proteins are found in E. coli but not B. subtilis (these regulatory proteins have been subject to a recent review [29]). DnaA, DnaD, and DnaB, together with the helicase loader DnaI (called DnaC in E. coli), the replicativ...

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Section: Membrane Attachmentmentioning

confidence: 81%

Chromosomal Replication Initiation Machinery of Low-G+C-Content Firmicutes

2012

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“…5 is broken by a random structure. This region is associated with phospholipids involved in rejuvenation of DnaA-ATP from DnaA-ADP (Garner et al, 1998) and consequently the dnaA1114 mutation may alter the balance between these two species of DnaA protein to favour the DnaA-ADP form, inactive for both autorepression and initiation.…”

Section: Chromosome Replication In the Dnaa(sx) Mutantsmentioning

confidence: 99%

Reduced initiation frequency from oriC restores viability of a temperature-sensitive Escherichia coli replisome mutant

Skovgaard

Løbner-Olesen

2005

Microbiology

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The dnaX gene of Escherichia coli encodes t and c clamp loader subunits of the replisome. Cells carrying the temperature-sensitive dnaX2016 mutation were induced for the SOS response at non-permissive temperature. The SOS induction most likely resulted from extensive replication fork collapse that exceeded the cells' capacity for restart. Seven mutations in the dnaA gene that partly suppressed the dnaX2016 temperature sensitivity were isolated and characterized. Each of the mutations caused a single amino acid change in domains III and IV of the DnaA protein, where nucleotide binding and DNA binding, respectively, reside. The diversity of dnaA(Sx) mutants obtained indicated that a direct interaction between the DnaA protein and t or c is unlikely and that the mechanism behind suppression is related to DnaA function. All dnaA(Sx) mutant cells were compromised for initiation of DNA replication, and contained fewer active replication forks than their wild-type counterparts. Conceivably, this led to a reduced number of replication fork collapses within each dnaX2016 dnaA(Sx) cell and prevented the SOS response. Lowered availability of wild-type DnaA protein also led to partial suppression of the dnaX2016 mutation, confirming that the dnaA(Sx) mode of suppression is indirect and results from a reduced initiation frequency at oriC.

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“…Amino acid sequence analysis of this region suggests the existence of an amphipathic helix (57), a motif commonly found in peripherally associated membrane proteins. Chemical crosslinking studies with a photoactivatable phospholipid analog demonstrated that the portion of DnaA centered about the putative amphipathic helix efficiently inserts into the hydrophobic interior of acidic phospholipid bilayers (16). A further indication that DnaA may have a membrane residency within cells is that partially purified DnaA protein is found aggregated in acidic phospholipid containing clusters (25,55), and the protein can be separated and rendered active for replication by treatment with phospholipase A 2 (25).…”

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confidence: 99%

DnaA, the Initiator of Escherichia coli Chromosomal Replication, Is Located at the Cell Membrane

Newman

Crooke

2000

J Bacteriol

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Given the lack of a nucleus in prokaryotic cells, the significance of spatial organization in bacterial chromosome replication is only beginning to be fully appreciated. DnaA protein, the initiator of chromosomal replication in Escherichia coli, is purified as a soluble protein, and in vitro it efficiently initiates replication of minichromosomes in membrane-free DNA synthesis reactions. However, its conversion from a replicatively inactive to an active form in vitro occurs through its association with acidic phospholipids in a lipid bilayer. To determine whether the in situ residence of DnaA protein is cytoplasmic, membrane associated, or both, we examined the cellular location of DnaA using immunogold cryothin-section electron microscopy and immunofluorescence. Both of these methods revealed that DnaA is localized at the cell membrane, further suggesting that initiation of chromosomal replication in E. coli is a membrane-affiliated event.Initiation of Escherichia coli chromosomal replication occurs at the chromosomal origin, oriC, a unique sequence that contains all necessary cis-acting elements (42, 47). To initiate a round of replication, approximately 20 molecules of DnaA protein bind to the DnaA boxes in oriC (10,13,14,38,41,62), promote strand opening of the AT-rich 13-mers (2,3,17), and facilitate the loading of DnaB helicase (14,40). The remaining components of the replisome are subsequently recruited to the sites of the newly formed replication forks (33,39).Given the central role of DnaA protein in the initiation process, its regulation is most likely crucial for normal, cellcycle-controlled chromosomal replication. While dnaA mRNA levels fluctuate throughout the cell cycle, DnaA protein levels remain relatively constant (51). Therefore, other mechanisms must regulate the protein's activity. Prominent among these in vitro is the influence on the replicative action of DnaA protein by the tight binding of ATP and ADP (52,65). Both the ADP and ATP forms of DnaA protein are capable of binding oriC (52). However, only the binding of ATP-DnaA leads to duplex melting and the ensuing initiation events (52,53,65). The ATP bound to DnaA protein is slowly hydrolyzed in a DNA-dependent manner, with the resulting ADP remaining tightly bound, thus producing inactive ADP-DnaA protein. A partially purified factor, termed IdaB, and ␤-subunit, the processivity factor of DNA polymerase III, stimulate the conversion of ATPDnaA to 29).Treatment of ADP-DnaA with anionic phospholipids causes the release of the bound nucleotide (8, 54). When exposed to anionic phospholipids in the presence of oriC DNA and excess ATP, replicatively inactive ADP-DnaA is rejuvenated into the active ATP form (9, 54). An examination of DnaA proteolytic fragments demonstrated that a distinct segment of DnaA is necessary for productive interaction with lipid bilayers (15). Amino acid sequence analysis of this region suggests the existence of an amphipathic helix (57), a motif commonly found in peripherally associated membrane proteins. Chemical cro...

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Membrane-mediated Release of Nucleotide from an Initiator of Chromosomal Replication, Escherichia coli DnaA, Occurs with Insertion of a Distinct Region of the Protein into the Lipid Bilayer

Cited by 60 publications

References 39 publications

Chromosomal Replication Initiation Machinery of Low-G+C-Content Firmicutes

Chromosomal Replication Initiation Machinery of Low-G+C-Content Firmicutes

Reduced initiation frequency from oriC restores viability of a temperature-sensitive Escherichia coli replisome mutant

DnaA, the Initiator of Escherichia coli Chromosomal Replication, Is Located at the Cell Membrane

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