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

Felczak, Magdalena M.; Sage, Jay M.; Hupert-Kocurek, Katarzyna; Aykul, Senem; Kaguni, Jon M.

doi:10.1074/jbc.m115.708586

Cited by 4 publications

(5 citation statements)

References 43 publications

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“…Proteins DnaB, DnaC, and the indicated mutants were purified essentially as described (10,56,73) and quantified by the dye-binding method (74) and also by SDS-PAGE after staining with Coomassie Blue using bovine serum albumin as a standard. DnaC⌬51 lacks the first 51 N-terminal residues of DnaC and fails to form the DnaB-DnaC complex due to its inability to interact with DnaB (56). Other replication proteins were purified as described (73,75).…”

Section: Methodsmentioning

confidence: 99%

“…3B). As a control, DnaB was incubated together with a truncated form of DnaC (DnaC⌬51), which lacks key residues within the first 51 N-terminal residues that interact directly with DnaB (33,56). Under conditions that would support assembly of the DnaB-DnaC complex (32), the presence of this mutant did not diminish the binding of DnaB to primase.…”

Section: Protein Dynamics Of Dnab Dnac and Primasementioning

confidence: 99%

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DnaC, the indispensable companion of DnaB helicase, controls the accessibility of DnaB helicase by primase

Felczak¹,

Chodavarapu²,

Kaguni

2017

Journal of Biological Chemistry

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Former studies relying on hydrogen/deuterium exchange analysis suggest that DnaC bound to DnaB alters the conformation of the N-terminal domain (NTD) of DnaB to impair the ability of this DNA helicase to interact with primase. Supporting this idea, the work described herein based on biosensor experiments and enzyme-linked immunosorbent assays shows that the DnaB-DnaC complex binds poorly to primase in comparison with DnaB alone. Using a structural model of DnaB complexed with the C-terminal domain of primase, we found that Ile-85 is located at the interface in the NTD of DnaB that contacts primase. An alanine substitution for Ile-85 specifically interfered with this interaction and impeded DnaB function in DNA replication, but not its activity as a DNA helicase or its ability to bind to ssDNA. By comparison, substitutions of Asn for Ile-136 (I136N) and Thr for Ile-142 (I142T) in a subdomain previously named the helical hairpin in the NTD of DnaB altered the conformation of the helical hairpin and/or compromised its pairwise arrangement with the companion subdomain in each brace of protomers of the DnaB hexamer. In contrast with the I85A mutant, the latter were defective in DNA replication due to impaired binding to both ssDNA and primase. In view of these findings, we propose that DnaC controls the ability of DnaB to interact with primase by modifying the conformation of the NTD of DnaB.

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Section: Methodsmentioning

confidence: 99%

Section: Protein Dynamics Of Dnab Dnac and Primasementioning

confidence: 99%

DnaC, the indispensable companion of DnaB helicase, controls the accessibility of DnaB helicase by primase

Felczak¹,

Chodavarapu²,

Kaguni

2017

Journal of Biological Chemistry

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“…Stimulated by ATP, DnaC is able to bind to single-stranded DNA, which is presumed to be required for its function in DNA replication [ 176 , 177 , 187 , 188 , 189 , 190 ]. Integrating these observations into the model described above, amino acid residues located in the inner channel of the DnaB-DnaC complex interact with the single-stranded DNA [ 177 ].…”

Section: Dnacmentioning

confidence: 99%

The Macromolecular Machines that Duplicate the Escherichia coli Chromosome as Targets for Drug Discovery

Kaguni

2018

Antibiotics

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DNA replication is an essential process. Although the fundamental strategies to duplicate chromosomes are similar in all free-living organisms, the enzymes of the three domains of life that perform similar functions in DNA replication differ in amino acid sequence and their three-dimensional structures. Moreover, the respective proteins generally utilize different enzymatic mechanisms. Hence, the replication proteins that are highly conserved among bacterial species are attractive targets to develop novel antibiotics as the compounds are unlikely to demonstrate off-target effects. For those proteins that differ among bacteria, compounds that are species-specific may be found. Escherichia coli has been developed as a model system to study DNA replication, serving as a benchmark for comparison. This review summarizes the functions of individual E. coli proteins, and the compounds that inhibit them.

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“…The ATPase activity of DnaC, a member of AAA+ proteins family, is not required for helicase hexamer opening and its loading by DnaC, hence the DnaB-binding domain of loader is sufficient for this process (Arias-Palomo et al, 2013 ). Yet the ATP hydrolysis by DnaC was proposed to occur during DnaB helicase activation, which results in DNA unwinding (Felczak et al, 2016 ).…”

Section: Helicase Loading Activation and Dna Unwindingmentioning

confidence: 99%

Replisome Assembly at Bacterial Chromosomes and Iteron Plasmids

Gross

Uciechowska

Konieczny

2016

Front. Mol. Biosci.

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The proper initiation and occurrence of DNA synthesis depends on the formation and rearrangements of nucleoprotein complexes within the origin of DNA replication. In this review article, we present the current knowledge on the molecular mechanism of replication complex assembly at the origin of bacterial chromosome and plasmid replicon containing direct repeats (iterons) within the origin sequence. We describe recent findings on chromosomal and plasmid replication initiators, DnaA and Rep proteins, respectively, and their sequence-specific interactions with double- and single-stranded DNA. Also, we discuss the current understanding of the activities of DnaA and Rep proteins required for replisome assembly that is fundamental to the duplication and stability of genetic information in bacterial cells.

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

Cited by 4 publications

References 43 publications

DnaC, the indispensable companion of DnaB helicase, controls the accessibility of DnaB helicase by primase

DnaC, the indispensable companion of DnaB helicase, controls the accessibility of DnaB helicase by primase

The Macromolecular Machines that Duplicate the Escherichia coli Chromosome as Targets for Drug Discovery

Replisome Assembly at Bacterial Chromosomes and Iteron Plasmids

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