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LC/MS label-free analysis of disulfide bond in C. bovis DnaA DI mutant S89C, Schumacher lab

ABSTRACT: Analysis of recombinant DnaA mutant construct with emphasis on identifying a natural disulfide occurence, detection done by crosslinking analysis via LC/MS-MS DDA on an Orbitrap Astral

INSTRUMENT(S):

ORGANISM(S): Bos Taurus (bovine)

SUBMITTER: Jeffrey Kuhn

LAB HEAD: Maria Schumacher

PROVIDER: PXD074582 | Pride | 2026-06-29

REPOSITORIES: Pride

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			Action	DRS
	ID136650_01_SPD60_OA10034_11063_091625.pdResult	Other
	ID136650_01_SPD60_OA10034_11063_091625.raw	Raw
	checksum.txt	Txt

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Publications

Structures of DnaA domain I reveal a dimer conserved across Actinomycetes.

Ellis Porter K PK Srinivasu Bindu Y BY Chavez Orozco Jose J Wray Gregory A GA Wales Thomas E TE Schumacher Maria A MA

Nucleic acids research 20260601 11

DNA replication is a fundamental process in biology, and initiation marks a key regulatory step. In bacteria, DNA replication is initiated by the DnaA protein. DnaA exhibits multidomain architecture, consisting of an N-terminal domain I, linker region, AAA+ family ATPase cassette, and C-terminal DNA-binding motif. Taxon-specific regulatory functions are primarily coordinated by the DnaA domain I (DnaADI), which exhibits substantial sequence variation across bacteria. Notably, although DnaADI has ...[more]

PMID: 42301917

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Multiple disulfide-bonded states confer conformational diversity in fibrinogen

Project description:Disulfide bonds constrain the polypeptide backbone and reduce conformational variability in proteins. The blood clotting protein fibrinogen is constitutively produced as multiple partially disulfide-bonded states, suggesting that individual fibrinogen molecules have a variety of conformational forms. This hypothesis was tested by resolving fibrinogen molecules on beads coated with different fibrinogen ligands and measuring their disulfide states by differential cysteine alkylation and mass spectrometry. Polyclonal anti-fibrinogen antibodies resolved states where all 11 measured disulfides across the molecule were significantly less formed. In contrast, the GHRP peptide, which binds the fibrinogen β-nodule, resolved states in which seven β-nodule and central E region disulfides were significantly more formed, while the GPRP peptide, which binds the γ-nodule, resolved states in which only one disulfide was significantly more formed. To probe the link between disulfide state and conformation, in silico analysis of all 32 possible disulfide-bonded states of the β-nodule revealed that the conformational flexibility of this domain and predicted GHRP interactions in its binding pocket are predicated on the oxidation state of one of the five β-nodule disulfides, βC424-βC437. These findings indicate that the different disulfide-bonded states of fibrinogen adopt an extensive array of conformations that are selectively recognized by different fibrinogen ligands.

2026-04-13 | PXD076459 | Pride

Anti-DnaA antibodies specificity validation by mass spectrometry

Project description:The ubiquitous protein DnaA is a chromosomal DNA replication initiator and transcription factor. It binds to 9-bp DNA sites called "DnaA-boxes" that are localized within the replication origin (oriC) and throughout chromosomal DNA, usually in the proximity of the promoter regions. During stress, bacteria undergo metabolic shifts that halt their growth and activate survival mechanisms. One outcome of these shifts is the accumulation of long chains of polyphosphate (polyP), an evolutionarily conserved linear polymer composed of up to 1,000 phosphate groups. It was previously identified that DnaA and the Lon protease bind to polyP, which stimulates Lon for the polyP-dependent DnaA proteolysis. We assumed that it have an impact not only on DnaA intracellular concentration but might also affect DNA binding pattern. To investigate this hypothesis and to analyze the DNA interaction profile of DnaA molecules remaining in stressed cells, we performed Chromatin Immunoprecipitation coupled with DNA sequencing (ChIP-seq). To exclude the presence of nonspecifically bound proteins in the sample after immunoprecipitation using anti-DnaA antibodies, mass spectrometry analysis was performed.

2026-03-02 | PXD061973 | Pride

Structures reveal DnaA domain I dimer conserved across Actinomycetes: implications for replication initiation

Project description:DNA replication is a fundamental process in biology with initiation marking its key, first step. In bacteria, DNA replication is initiated by the DnaA protein. DnaA exhibits multidomain architecture, consisting of an N-terminal domain I, linker region, AAA+ family ATPase cassette, and C-terminal DNA-binding motif. Taxon-specific regulatory functions are primarily coordinated by the DnaA domain I, which exhibits substantial sequence variation across bacteria. Notably, although the DnaA domain I has been shown to be essential, its contributions to initiation are not completely understood. Importantly, studies have indicated a role for DnaA domain I dimerization in the cooperative assembly of the initiation complex at the origin. However, the mechanism(s) and molecular basis of DnaA domain I dimerization have proved elusive. Here, we report structures of the DnaA domain I from ten Actinobacterial species. Strikingly, all structures reveal the same, unique dimer, and key elements which support DnaA domain I self-interaction are broadly conserved across the class Actinomycetes. Further, a suite of biochemical oligomerization assays and HDX-MS studies support the structural dimer. These findings suggest weak dimerization, as mediated by the DnaA domain I, acts a fine-tuned trigger in cooperative oriC assembly and that this is a broadly conserved biological mechanism for replication initiation in the class Actinomycetes.

2026-05-27 | PXD074705 | Pride

Expression profiles of dnaA null, rok null, and dnaA null; rok null mutants

2015-06-13 | GSE69788 | GEO

ChIP-seq experiment: DnaA binding to E. coli chromosome before stress, during stress, and stress recovery

Project description:The ubiquitous protein DnaA is a chromosomal DNA replication initiator and transcription factor. It binds to 9-bp DNA sites called \"DnaA-boxes\" that are localized within the replication origin (oriC) and throughout chromosomal DNA, usually in the proximity of the promoter regions. During stress, bacteria undergo metabolic shifts that halt their growth and activate survival mechanisms. One outcome of these shifts is the accumulation of long chains of polyphosphate (polyP), an evolutionarily conserved linear polymer composed of up to 1,000 phosphate groups. It was previously identified that DnaA and the Lon protease bind to polyP, which stimulates Lon for the polyP-dependent DnaA proteolysis. We assumed that it have an impact not only on DnaA intracellular concentration but might also affect DNA binding pattern. To investigate this hypothesis and to analyze the DNA interaction profile of DnaA molecules remaining in stressed cells, we performed Chromatin Immunoprecipitation coupled with DNA sequencing (ChIP-seq).

2026-03-13 | E-MTAB-14964 | biostudies-arrayexpress

Label-free protein quantification in freshly ejaculated versus post-mating spermatophores of the noble crayfish Astacus astacus

Project description:Upon completion of spermatogenesis in crayfish testis, spermatozoa enter the vasa deferentia and are packaged into spermatophores that apparently function in the transfer of spermatozoa from male to the female during mating. In Astacidae and Parastacidae, spermatophores are deposited on the ventral surface of the female, where they remain for several days before initiation of ovulation and subsequent fertilization. This is accompanied by spermatophore morphological changes, suggesting that final maturation of decapod spermatozoa takes place in the seminal receptacle of the female

2015-04-13 | PXD001929 | Pride

Regulatory elements coordinating initiation of chromosome replication to the Escherichia coli cell cycle

Project description:Escherichia coli coordinates replication and division cycles by initiating replication at a narrow range of cell sizes. By tracking replisomes in individual cells through thousands of division cycles in wild-type and mutant strains, we were able to compare the relative importance of previously described control systems. We found that accurate triggering of initiation does not require synthesis of new DnaA. The initiation size increased only marginally as DnaA was diluted by growth after dnaA expression had been turned off. This suggests that the conversion of DnaA between its active ATP- and inactive ADP-bound states is more important for initiation size control than the total free concentration of DnaA. In addition, we found that the known ATP/ADP-converters DARS and datA compensate for each other, although the removal of them makes the initiation size more sensitive to the concentration of DnaA. Only disruption of the regulatory inactivation of DnaA (RIDA) mechanism had a radical impact on replication initiation. This result was corroborated by the finding that termination of one round of replication correlates with the next initiation at intermediate growth rates, as would be the case if RIDA-mediated conversion from DnaA-ATP to DnaA-ADP abruptly stops at termination and DnaA-ATP starts accumulating.

2023-05-12 | PXD036580 | Pride

The Escherichia coli replication initiator DnaA is titrated on the chromosome

Project description:DNA replication initiation is orchestrated in bacteria by the replication initiator DnaA. Two models for regulation of DnaA activity in Escherichia coli have been proposed: the switch between an active and inactive form and the titration of DnaA on the chromosome. Although proposed decades ago, experimental evidence of a titration-based control mechanism is still lacking. Here, we first identified a conserved high-density region of binding motifs near the origin of replication, an advantageous trait for titration of DnaA. We then investigated the mobility of DnaA by visualising single proteins inside single cells of wild-type and deletion mutants E. coli strains, while monitoring cellular size and DNA content. Our results indicate that the chromosome of E. coli controls the free amount of DnaA in a growth rate-dependent fashion. Moreover, they address long-standing questions on the relevance of DnaA titration in stabilising DNA replication by preventing re-initiation events during slow growth.

2025-07-28 | PXD064841 | Pride

Genome-wide binding of replication initiation proteins in Bacillus subtilis

Project description:Initiation of DNA replication requires binding of the initiator protein, DnaA, to specific binding sites in the chromosomal origin of replication, oriC. In low G+C Gram-positive bacteria, the primosomal proteins DnaD and DnaB, in conjunction with loader ATPase DnaI, load the replicative helicase at oriC, and this depends on DnaA. DnaD and DnaB are also required to load the replicative helicase outside of oriC during replication restart, in a DnaA-independent manner. DnaA also binds to many sites around the chromosome, outside of oriC, and acts as a transcription factor at several of these. Using chromatin immunoprecipitation, we found that DnaD and DnaB, but not the replicative helicase, are associated with many of the chromosomal regions bound by DnaA in vivo in Bacillus subtilis. This association was dependent on DnaA and the order of recruitment was the same as that at oriC, but was independent of a functional oriC. The presence of DnaD and DnaB at the secondary (non-oriC) targets of DnaA in the absence of helicase loading indicates a possible role for DnaD and DnaB in modulating the activity of DnaA.

2010-12-07 | GSE23686 | GEO

Expression profiles of dnaA null, rok null, and dnaA null; rok null mutants

Project description:To identify potential targets of co-regulation by DnaA and Rok, we compared the transcriptional profiles of dnaA null, rok null, and dnaA null rok null mutants. Because a dnaA null mutant requires an oriC- strain background, we used an oriC- oriN+ background for all strains, to allow direct comparisons. We compared biological triplicates for each of the following strains: dnaA null (CAL2074), rok null (CAS196), dnaA null rok null (CAS192), and dnaA+ rok+ (CAL2083; control).

2015-06-13 | E-GEOD-69788 | biostudies-arrayexpress

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