DnaC traps DnaB as an open ring and remodels the domain that binds primase

Chodavarapu, Sundari; Jones, A. Daniel; Feig, Michael; Kaguni, Jon M.

doi:10.1093/nar/gkv961

Cited by 31 publications

(51 citation statements)

References 67 publications

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“…The Mutant DnaCs Are Able to Interact with DnaB-Studies indicate that two sites near the N terminus of DnaC interact with DnaB in the DnaB-DnaC complex (10,28). Hence, we expected that the substitutions would not cause impaired binding to DnaB because they do not reside in these sites.…”

Section: Resultsmentioning

confidence: 99%

“…11 and 17. The residues shaded in gray represent segments that bind to DnaB (28). Those shaded in blue correspond to the initiator specific motif (ISM; residues 136 -162) (12).…”

Section: Resultsmentioning

confidence: 99%

“…ATP also increases the affinity of DnaC for ssDNA, but the loop is not an obligate requirement as the affinity of DnaC for ssDNA only increases 2-3-fold (13,19). For E. coli DnaC, Arg 236 corresponds to the Sensor II residue; Phe 231 and Trp 233 reside in the loop in a homology model of E. coli DnaC constructed using the above x-ray structure of A. aeolicus DnaC as a template (28).…”

Section: F231s and W233l Are Thermolabile At The Step Of Loading Of Tmentioning

confidence: 99%

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Substitutions of Conserved Residues in the C-terminal Region of DnaC Cause Thermolability in Helicase Loading

Felczak

Sage

Hupert-Kocurek

et al. 2016

Journal of Biological Chemistry

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The DnaB-DnaC complex binds to the unwound DNA within the Escherichia coli replication origin in the helicase loading process, but the biochemical events that lead to its stable binding are uncertain. This study characterizes the function of specific C-terminal residues of DnaC. Genetic and biochemical characterization of proteins bearing F231S and W233L substitutions of DnaC reveals that their activity is thermolabile.

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Section: Resultsmentioning

confidence: 99%

“…11 and 17. The residues shaded in gray represent segments that bind to DnaB (28). Those shaded in blue correspond to the initiator specific motif (ISM; residues 136 -162) (12).…”

Section: Resultsmentioning

confidence: 99%

Section: F231s and W233l Are Thermolabile At The Step Of Loading Of Tmentioning

confidence: 99%

See 1 more Smart Citation

Substitutions of Conserved Residues in the C-terminal Region of DnaC Cause Thermolability in Helicase Loading

Felczak

Sage

Hupert-Kocurek

et al. 2016

Journal of Biological Chemistry

Self Cite

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“…Genetic and biochemical studies demonstrated that amino acid substitutions within residues 8–11 and 31–44 of DnaC abrogate its interaction with DnaB and the function of DnaC in replication initiation [140]. In hydrogen-deuterium exchange experiments, which measures protein dynamics by the relative ability of amide hydrogens in a protein to exchange with deuterium in a buffer made with heavy water, peptides corresponding to these segments of DnaC were found to become occluded when DnaC is complexed to DnaB [141]. Together, these results indicate that these regions of DnaC interact directly with DnaB.…”

Section: Mechanism Of Initiationmentioning

confidence: 99%

“…Other evidence showed that mutant DnaBs bearing I297A, L304A, or E435A substitutions are defective in complementing a temperature-sensitive dnaB mutant at nonpermissive temperature [141]. To substantiate that the E435A substitution interferes with DnaB function, biochemical experiments were performed, which showed that this protein is inactive in DNA replication of an oriC -containing plasmid.…”

Section: Mechanism Of Initiationmentioning

confidence: 99%

Replication Initiation in Bacteria

Chodavarapu

Kaguni

2016

The Enzymes

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The initiation of chromosomal DNA replication starts at a replication origin, which in bacteria is a discrete locus that contains DNA sequence motifs recognized by an initiator protein whose role is to assemble the replication fork machinery at this site. In bacteria with a single chromosome, DnaA is the initiator and is highly conserved in all bacteria. As an adenine nucleotide binding protein, DnaA bound to ATP is active in the assembly of a DnaA oligomer onto these sites. Other proteins modulate DnaA oligomerization via their interaction with the N-terminal region of DnaA. Following the DnaA-dependent unwinding of an AT-rich region within the replication origin, DnaA then mediates the binding of DnaB, the replicative DNA helicase, in a complex with DnaC to form an intermediate named the prepriming complex. In the formation of this intermediate, the helicase is loaded onto the unwound region within the replication origin. As DnaC bound to DnaB inhibits its activity as a DNA helicase, DnaC must dissociate to activate DnaB. Apparently, the interaction of DnaB with primase (DnaG) and primer formation leads to the release of DnaC from DnaB, which is coordinated with or followed by translocation of DnaB to the junction of the replication fork. There, DnaB is able to coordinate its activity as a DNA helicase with the cellular replicase, DNA polymerase III holoenzyme, which uses the primers made by primase for leading strand DNA synthesis.

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Assessing heterogeneity in oligomeric AAA+ machines

Sysoeva

2016

Cell. Mol. Life Sci.

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ATPases Associated with various cellular Activities (AAA+ ATPases) are molecular motors that use the energy of ATP binding and hydrolysis to remodel their target macromolecules. The majority of these ATPases form ring-shaped hexamers in which the active sites are located at the interfaces between neighboring subunits. Structural changes initiate in an active site and propagate to distant motor parts that interface and reshape the target macromolecules, thereby performing mechanical work. During the functioning cycle, the AAA+ motor transits through multiple distinct states. Ring architecture and placement of the catalytic sites at the intersubunit interfaces allow for a unique level of coordination among subunits of the motor. This in turn results in conformational differences among subunits and overall asymmetry of the motor ring as it functions. To date, a large amount of structural information has been gathered for different AAA+ motors, but even for the most characterized of them only a few structural states are known and the full mechanistic cycle cannot be yet reconstructed. Therefore, the first part of this work will provide a broad overview of what arrangements of AAA+ subunits have been structurally observed focusing on diversity of ATPase oligomeric ensembles and heterogeneity within the ensembles. The second part of this review will concentrate on methods that assess structural and functional heterogeneity among subunits of AAA+ motors, thus bringing us closer to understanding the mechanism of these fascinating molecular motors.

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DnaC traps DnaB as an open ring and remodels the domain that binds primase

Cited by 31 publications

References 67 publications

Substitutions of Conserved Residues in the C-terminal Region of DnaC Cause Thermolability in Helicase Loading

Substitutions of Conserved Residues in the C-terminal Region of DnaC Cause Thermolability in Helicase Loading

Replication Initiation in Bacteria

Assessing heterogeneity in oligomeric AAA+ machines

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