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1.
J Biol Chem ; 300(5): 107275, 2024 May.
Article in English | MEDLINE | ID: mdl-38588814

ABSTRACT

DNA replication in Escherichia coli starts with loading of the replicative helicase, DnaB, onto DNA. This reaction requires the DnaC loader protein, which forms a 6:6 complex with DnaB and opens a channel in the DnaB hexamer through which single-stranded DNA is thought to pass. During replication, replisomes frequently encounter DNA damage and nucleoprotein complexes that can lead to replication fork collapse. Such events require DnaB re-loading onto DNA to allow replication to continue. Replication restart proteins mediate this process by recruiting DnaB6/DnaC6 to abandoned DNA replication forks. Several dnaC mutations that bypass the requirement for replication restart proteins or that block replication restart have been identified in E. coli. To better understand how these DnaC variants function, we have purified and characterized the protein products of several such alleles. Unlike wild-type DnaC, three of the variants (DnaC 809, DnaC 809,820, and DnaC 811) can load DnaB onto replication forks bound by single-stranded DNA-binding protein. DnaC 809 can also load DnaB onto double-stranded DNA. These results suggest that structural changes in the variant DnaB6/DnaC6 complexes expand the range of DNA substrates that can be used for DnaB loading, obviating the need for the existing replication restart pathways. The protein product of dnaC1331, which phenocopies deletion of the priB replication restart gene, blocks loading through the major restart pathway in vitro. Overall, the results of our study highlight the utility of bacterial DnaC variants as tools for probing the regulatory mechanisms that govern replicative helicase loading.


Subject(s)
DNA Replication , DnaB Helicases , Escherichia coli Proteins , Escherichia coli , Escherichia coli Proteins/metabolism , Escherichia coli Proteins/genetics , Escherichia coli Proteins/chemistry , Escherichia coli/metabolism , Escherichia coli/genetics , DnaB Helicases/metabolism , DnaB Helicases/genetics , DnaB Helicases/chemistry , DNA, Bacterial/metabolism , DNA, Bacterial/genetics , DNA, Single-Stranded/metabolism , DNA, Single-Stranded/genetics , Mutation
2.
Proc Natl Acad Sci U S A ; 121(19): e2317954121, 2024 May 07.
Article in English | MEDLINE | ID: mdl-38683976

ABSTRACT

Several microbial genomes lack textbook-defined essential genes. If an essential gene is absent from a genome, then an evolutionarily independent gene of unknown function complements its function. Here, we identified frequent nonhomologous replacement of an essential component of DNA replication initiation, a replicative helicase loader gene, in Vibrionaceae. Our analysis of Vibrionaceae genomes revealed two genes with unknown function, named vdhL1 and vdhL2, that were substantially enriched in genomes without the known helicase-loader genes. These genes showed no sequence similarities to genes with known function but encoded proteins structurally similar with a viral helicase loader. Analyses of genomic syntenies and coevolution with helicase genes suggested that vdhL1/2 encodes a helicase loader. The in vitro assay showed that Vibrio harveyi VdhL1 and Vibrio ezurae VdhL2 promote the helicase activity of DnaB. Furthermore, molecular phylogenetics suggested that vdhL1/2 were derived from phages and replaced an intrinsic helicase loader gene of Vibrionaceae over 20 times. This high replacement frequency implies the host's advantage in acquiring a viral helicase loader gene.


Subject(s)
DNA Helicases , DNA Replication , Phylogeny , Vibrionaceae , Vibrionaceae/genetics , Vibrionaceae/enzymology , DNA Helicases/metabolism , DNA Helicases/genetics , Bacterial Proteins/metabolism , Bacterial Proteins/genetics , Viral Proteins/genetics , Viral Proteins/metabolism , Bacteriophages/genetics , Bacteriophages/enzymology , Evolution, Molecular , Genome, Bacterial , DnaB Helicases/metabolism , DnaB Helicases/genetics , Vibrio/genetics , Vibrio/enzymology
3.
Acta Crystallogr D Struct Biol ; 79(Pt 2): 177-187, 2023 Feb 01.
Article in English | MEDLINE | ID: mdl-36762863

ABSTRACT

During the initiation step of bacterial genome replication, replicative helicases depend on specialized proteins for their loading onto oriC. DnaC and DnaI were the first loaders to be characterized. However, most bacteria do not contain any of these genes, which are domesticated phage elements that have replaced the ancestral and unrelated loader gene dciA several times during evolution. To understand how DciA assists the loading of DnaB, the crystal structure of the complex from Vibrio cholerae was determined, in which two VcDciA molecules interact with a dimer of VcDnaB without changing its canonical structure. The data showed that the VcDciA binding site on VcDnaB is the conserved module formed by the linker helix LH of one monomer and the determinant helix DH of the second monomer. Interestingly, DnaC from Escherichia coli also targets this module onto EcDnaB. Thanks to their common target site, it was shown that VcDciA and EcDnaC could be functionally interchanged in vitro despite sharing no structural similarity. This represents a milestone in understanding the mechanism employed by phage helicase loaders to hijack bacterial replicative helicases during evolution.


Subject(s)
Escherichia coli Proteins , Escherichia coli Proteins/chemistry , DNA Replication , DnaB Helicases/chemistry , DnaB Helicases/genetics , DnaB Helicases/metabolism , DNA Helicases/chemistry , Bacteria/metabolism , Escherichia coli/genetics , Binding Sites , Bacterial Proteins/chemistry
4.
Nucleic Acids Res ; 51(9): 4302-4321, 2023 05 22.
Article in English | MEDLINE | ID: mdl-36416272

ABSTRACT

Bidirectional DNA replication from a chromosome origin requires the asymmetric loading of two helicases, one for each replisome. Our understanding of the molecular mechanisms underpinning helicase loading at bacterial chromosome origins is incomplete. Here we report both positive and negative mechanisms for directing helicase recruitment in the model organism Bacillus subtilis. Systematic characterization of the essential initiation protein DnaD revealed distinct protein interfaces required for homo-oligomerization, interaction with the master initiator protein DnaA, and interaction with the helicase co-loader protein DnaB. Informed by these properties of DnaD, we went on to find that the developmentally expressed repressor of DNA replication initiation, SirA, blocks the interaction between DnaD and DnaA, thereby restricting helicase recruitment from the origin during sporulation to inhibit further initiation events. These results advance our understanding of the mechanisms underpinning DNA replication initiation in B. subtilis, as well as guiding the search for essential cellular activities to target for antimicrobial drug design.


Subject(s)
Bacillus subtilis , Bacterial Proteins , DNA Helicases , Spores, Bacterial , Bacillus subtilis/genetics , Bacillus subtilis/metabolism , Bacillus subtilis/physiology , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , DNA Helicases/genetics , DNA Helicases/metabolism , DNA Replication , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , DnaB Helicases/genetics , DnaB Helicases/metabolism , Replication Origin , Spores, Bacterial/metabolism
5.
Protein Pept Lett ; 30(1): 35-43, 2023.
Article in English | MEDLINE | ID: mdl-36336812

ABSTRACT

BACKGROUND: DNA helicases are unwinding enzymes that are essential for many cellular processes. Research has suggested that both the model microorganisms of a single chromosome and the model microorganisms of multiple chromosomes adopt DNA helicases encoded by chromosome I. Therefore, studying DNA helicases encoded by chromosome II may lay some foundation for understanding nucleic acid metabolism processes. OBJECTIVE: To prove the existence of DNA helicase encoded by chromosome II and to reveal its difference compared to DNA helicase encoded by chromosome I. METHODS: The DNA helicases of Pseudoalteromonas spongiae JCM 12884T and Pseudoalteromonas tunicata DSM 14096T were analyzed by sequence alignment and phylogenetic relationships with other known DNA helicases. Then, proteins of P. spongiae JCM 12884T and P. tunicata DSM 14096T were obtained by heterologous expression. N-terminal sequencing and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis were performed to confirm the form of proteins. A fluorescence resonance energy transfer (FRET) assay was used to measure the activity of helicases. RESULTS: DnaB-pspo and DnaB-ptun belong to the same family, the PRK08840 superfamily, and form a branch with helicases encoded by chromosome I. YwqA-pspo and YwqA-ptun have similar domains and form another branch with helicases encoded by chromosome II. All four helicases have DNA unwinding activity. YwqA is more efficient than DnaB for DNA unwinding, especially YwqA-pspo, which is encoded by bidirectional replication chromosome II. CONCLUSION: This is the first study to show that the existence of a DNA helicase encoded by chromosome II, and DNA helicase encoded by chromosome II is more efficient than chromosome I for DNA unwinding.


Subject(s)
Escherichia coli , Tandem Mass Spectrometry , DnaB Helicases/chemistry , DnaB Helicases/genetics , DnaB Helicases/metabolism , Chromatography, Liquid , Phylogeny , Escherichia coli/genetics , DNA/chemistry , DNA Helicases/genetics , DNA Helicases/metabolism , DNA Replication , Bacterial Proteins/chemistry
6.
Nucleic Acids Res ; 50(22): 12896-12912, 2022 12 09.
Article in English | MEDLINE | ID: mdl-36484102

ABSTRACT

The replicative DNA helicase translocates on single-stranded DNA to drive replication forks during chromosome replication. In most bacteria the ubiquitous replicative helicase, DnaB, co-evolved with the accessory subunit DciA, but how they function remains incompletely understood. Here, using the model bacterium Caulobacter crescentus, we demonstrate that DciA plays a prominent role in DNA replication fork maintenance. Cell cycle analyses using a synchronized Caulobacter cell population showed that cells devoid of DciA exhibit a severe delay in fork progression. Biochemical characterization revealed that the DnaB helicase in its default state forms a hexamer that inhibits self-loading onto single-stranded DNA. We found that upon binding to DciA, the DnaB hexamer undergoes conformational changes required for encircling single-stranded DNA, thereby establishing the replication fork. Further investigation of the functional structure of DciA revealed that the C-terminus of DciA includes conserved leucine residues responsible for DnaB binding and is essential for DciA in vivo functions. We propose that DciA stimulates loading of DnaB onto single strands through topological isomerization of the DnaB structure, thereby ensuring fork progression. Given that the DnaB-DciA modules are widespread among eubacterial species, our findings suggest that a common mechanism underlies chromosome replication.


Subject(s)
Bacterial Proteins , Caulobacter crescentus , Chromosomes, Bacterial , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Caulobacter crescentus/genetics , Caulobacter crescentus/metabolism , DNA Replication/genetics , DNA, Single-Stranded/metabolism , DnaB Helicases/metabolism , Chromosomes, Bacterial/genetics , Chromosomes, Bacterial/metabolism
7.
Methods Enzymol ; 672: 125-142, 2022.
Article in English | MEDLINE | ID: mdl-35934472

ABSTRACT

The genome of prokaryotes can be damaged by a variety of endogenous and exogenous factors, including reactive oxygen species, UV exposure, and antibiotics. To better understand these repair processes and the impact they may have on DNA replication, normal genome maintenance processes can be perturbed by removing or editing associated genes and monitoring DNA repair outcomes. In particular, the replisome activities of DNA unwinding by the helicase and DNA synthesis by the polymerase must be tightly coupled to prevent any appreciable single strand DNA (ssDNA) from accumulating and amplifying genomic stress. If decoupled, vulnerable ssDNA would persist, likely leading to double strand breaks (DSBs) or requiring replication restart mechanisms downstream of a stall. In either case, free 3'-OH strands would exist, resulting from ssDNA gaps in the leading strand or complete DSBs. Terminal deoxyribonucleotide transferase (TdT)-mediated dUTP nick end labeling (TUNEL) can enzymatically label ssDNA ends with bromo-deoxy uridine triphosphate (BrdU) to detect free 3'-OH DNA ends in the E. coli genome. Labeled DNA ends can be detected and quantified using fluorescence microscopy or flow cytometry. This methodology is useful in applications where in situ investigation of DNA damage and repair are of interest, including effects from enzyme mutations or deletions and exposure to various environmental conditions.


Subject(s)
DNA, Single-Stranded , Escherichia coli , DNA , DNA Nucleotidylexotransferase , DNA, Single-Stranded/genetics , DNA-Directed DNA Polymerase/metabolism , DnaB Helicases/genetics , Escherichia coli/metabolism , In Situ Nick-End Labeling
8.
Methods Enzymol ; 672: 143-152, 2022.
Article in English | MEDLINE | ID: mdl-35934473

ABSTRACT

Ring-shaped hexameric helicases are an essential class of enzymes that unwind duplex nucleic acids to support a variety of cellular processes. Because of their critical roles in cells, hexameric helicase dysfunction has been linked to DNA damage and genomic instability. Biochemical characterization of hexameric helicase activity and regulation in vitro is necessary for understanding enzyme function and aiding drug discovery efforts. In this chapter, we describe protocols for characterizing mechanisms of helicase loading, activation, and unwinding using the model replicative hexameric DnaB helicase and its cognate DnaC loading factor from E. coli.


Subject(s)
Escherichia coli Proteins , Escherichia coli , Bacterial Proteins/genetics , DNA Helicases/chemistry , DNA Replication , DnaB Helicases/chemistry , DnaB Helicases/genetics , DnaB Helicases/metabolism , Escherichia coli Proteins/chemistry
9.
FEBS Lett ; 596(16): 2031-2040, 2022 08.
Article in English | MEDLINE | ID: mdl-35568982

ABSTRACT

To enable chromosomal replication, DNA is unwound by the ATPase molecular motor replicative helicase. The bacterial helicase DnaB is a ring-shaped homo-hexamer whose conformational dynamics are being studied through its different 3D structural states adopted along its functional cycle. Our findings describe a new crystal structure for the apo-DnaB from Vibrio cholerae, forming a planar hexamer with pseudo-symmetry, constituted by a trimer of dimers in which the C-terminal domains delimit a triskelion-shaped hole. This hexamer is labile and inactive. We suggest that it represents an intermediate state allowing the formation of the active NTP-bound hexamer from dimers.


Subject(s)
Vibrio cholerae , Bacterial Proteins , DNA Helicases , DNA Replication , DnaB Helicases , Escherichia coli , Protein Multimerization
10.
J Biol Chem ; 298(6): 102051, 2022 06.
Article in English | MEDLINE | ID: mdl-35598828

ABSTRACT

Unwinding of the replication origin and loading of DNA helicases underlie the initiation of chromosomal replication. In Escherichia coli, the minimal origin oriC contains a duplex unwinding element (DUE) region and three (Left, Middle, and Right) regions that bind the initiator protein DnaA. The Left/Right regions bear a set of DnaA-binding sequences, constituting the Left/Right-DnaA subcomplexes, while the Middle region has a single DnaA-binding site, which stimulates formation of the Left/Right-DnaA subcomplexes. In addition, a DUE-flanking AT-cluster element (TATTAAAAAGAA) is located just outside of the minimal oriC region. The Left-DnaA subcomplex promotes unwinding of the flanking DUE exposing TT[A/G]T(T) sequences that then bind to the Left-DnaA subcomplex, stabilizing the unwound state required for DnaB helicase loading. However, the role of the Right-DnaA subcomplex is largely unclear. Here, we show that DUE unwinding by both the Left/Right-DnaA subcomplexes, but not the Left-DnaA subcomplex only, was stimulated by a DUE-terminal subregion flanking the AT-cluster. Consistently, we found the Right-DnaA subcomplex-bound single-stranded DUE and AT-cluster regions. In addition, the Left/Right-DnaA subcomplexes bound DnaB helicase independently. For only the Left-DnaA subcomplex, we show the AT-cluster was crucial for DnaB loading. The role of unwound DNA binding of the Right-DnaA subcomplex was further supported by in vivo data. Taken together, we propose a model in which the Right-DnaA subcomplex dynamically interacts with the unwound DUE, assisting in DUE unwinding and efficient loading of DnaB helicases, while in the absence of the Right-DnaA subcomplex, the AT-cluster assists in those processes, supporting robustness of replication initiation.


Subject(s)
Bacterial Proteins , DNA-Binding Proteins , DnaB Helicases , Origin Recognition Complex , Replication Origin , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , DNA Replication , DNA, Bacterial/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , DnaB Helicases/genetics , DnaB Helicases/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Origin Recognition Complex/genetics
11.
J Biochem ; 171(6): 605-607, 2022 May 27.
Article in English | MEDLINE | ID: mdl-35238386

ABSTRACT

DNA replication is an essential, precisely regulated process that occurs once in a cell cycle. In the Gram-negative bacterium Escherichia coli, the replicative helicase EcDnaB and the helicase loader EcDnaC play key roles in the initiation step at the replication origin, oriC. EcDnaB and EcDnaC form a heterododecamer, in which hexameric EcDnaB is bound to hexameric EcDnaC. Using genetic, biochemical and structural biology approaches, many groups have probed the mechanism of replicative helicase loading, using helicases and helicase loaders from various species. Recent X-ray crystallography and cryogenic electron microscopy (cryo-EM) structural studies of the EcDnaB-EcDnaC complex revealed that the interaction of DnaC with DnaB triggers distortion accumulation on the closed ring of hexameric DnaB, inducing DnaB subunits to adopt the open helical form for replication progression. The high-resolution crystal structure of the DnaB-DnaC complex solved by Nagata et al. contributed to a better understanding of the conformational rearrangement of the DnaB ring. In addition to the structural alterations in DnaB subunits by DnaC, the binding of single-stranded DNA (ssDNA) substrates alters the ATP- and ADP-bound forms of DnaB and DnaC. These studies have proposed mechanisms by which DnaC regulates helicase loading onto ssDNA.


Subject(s)
Escherichia coli Proteins , Escherichia coli , Bacterial Proteins/metabolism , DNA Helicases/metabolism , DNA Replication , DNA, Single-Stranded , DnaB Helicases/chemistry , Escherichia coli/metabolism , Escherichia coli Proteins/metabolism
12.
Trends Biochem Sci ; 47(7): 620-630, 2022 07.
Article in English | MEDLINE | ID: mdl-35351361

ABSTRACT

Dedicated loader proteins play essential roles in bacterial DNA replication by opening ring-shaped DnaB-family helicases and chaperoning single-stranded (ss)DNA into a central motor chamber as a prelude to DNA unwinding. Although unrelated in sequence, the Escherichia coli DnaC and bacteriophage λ P loaders feature a similar overall architecture: a globular domain linked to an extended lasso/grappling hook element, located at their N and C termini, respectively. Both loaders remodel a closed DnaB ring into nearly identical right-handed open conformations. The sole element shared by the loaders is a single alpha helix, which binds to the same site on the helicase. Physical features of the loaders establish that DnaC and λ P evolved independently to converge, through molecular mimicry, on a common helicase-opening mechanism.


Subject(s)
Escherichia coli Proteins , Bacterial Proteins/metabolism , DNA Helicases/metabolism , DNA Replication , DNA, Single-Stranded , DnaB Helicases/chemistry , DnaB Helicases/genetics , DnaB Helicases/metabolism , Escherichia coli/metabolism , Escherichia coli Proteins/chemistry
13.
PLoS Genet ; 17(11): e1009886, 2021 11.
Article in English | MEDLINE | ID: mdl-34767550

ABSTRACT

Helicase regulation involves modulation of unwinding speed to maintain coordination of DNA replication fork activities and is vital for replisome progression. Currently, mechanisms for helicase regulation that involve interactions with both DNA strands through a steric exclusion and wrapping (SEW) model and conformational shifts between dilated and constricted states have been examined in vitro. To better understand the mechanism and cellular impact of helicase regulation, we used CRISPR-Cas9 genome editing to study four previously identified SEW-deficient mutants of the bacterial replicative helicase DnaB. We discovered that these four SEW mutations stabilize constricted states, with more fully constricted mutants having a generally greater impact on genomic stress, suggesting a dynamic model for helicase regulation that involves both excluded strand interactions and conformational states. These dnaB mutations result in increased chromosome complexities, less stable genomes, and ultimately less viable and fit strains. Specifically, dnaB:mut strains present with increased mutational frequencies without significantly inducing SOS, consistent with leaving single-strand gaps in the genome during replication that are subsequently filled with lower fidelity. This work explores the genomic impacts of helicase dysregulation in vivo, supporting a combined dynamic regulatory mechanism involving a spectrum of DnaB conformational changes and relates current mechanistic understanding to functional helicase behavior at the replication fork.


Subject(s)
Chromosomes, Bacterial , DnaB Helicases/metabolism , Escherichia coli/genetics , Genomic Instability , CRISPR-Cas Systems , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , DnaB Helicases/chemistry , DnaB Helicases/genetics , Escherichia coli/enzymology , Mutation
14.
J Magn Reson ; 332: 107075, 2021 11.
Article in English | MEDLINE | ID: mdl-34597956

ABSTRACT

Labeling of biomolecules with a paramagnetic probe for nuclear magnetic resonance (NMR) spectroscopy enables determining long-range distance restraints, which are otherwise not accessible by classically used dipolar coupling-based NMR approaches. Distance restraints derived from paramagnetic relaxation enhancements (PREs) can facilitate the structure determination of large proteins and protein complexes. We herein present the site-directed labeling of the large oligomeric bacterial DnaB helicase from Helicobacter pylori with cysteine-reactive maleimide tags carrying either a nitroxide radical or a lanthanide ion. The success of the labeling reaction was followed by quantitative continuous-wave electron paramagnetic resonance (EPR) experiments performed on the nitroxide-labeled protein. PREs were extracted site-specifically from 2D and 3D solid-state NMR spectra. A good agreement with predicted PRE values, derived by computational modeling of nitroxide and Gd3+ tags in the low-resolution DnaB crystal structure, was found. Comparison of experimental PREs and model-predicted spin label-nucleus distances indicated that the size of the "blind sphere" around the paramagnetic center, in which NMR resonances are not detected, is slightly larger for Gd3+ (∼14 Å) than for nitroxide (∼11 Å) in 13C-detected 2D spectra of DnaB. We also present Gd3+-Gd3+ dipolar electron-electron resonance EPR experiments on DnaB supporting the conclusion that DnaB was present as a hexameric assembly.


Subject(s)
Proteins , DnaB Helicases , Electron Spin Resonance Spectroscopy , Magnetic Resonance Spectroscopy , Spin Labels
15.
Nat Commun ; 12(1): 5293, 2021 09 06.
Article in English | MEDLINE | ID: mdl-34489448

ABSTRACT

The ATP hydrolysis transition state of motor proteins is a weakly populated protein state that can be stabilized and investigated by replacing ATP with chemical mimics. We present atomic-level structural and dynamic insights on a state created by ADP aluminum fluoride binding to the bacterial DnaB helicase from Helicobacter pylori. We determined the positioning of the metal ion cofactor within the active site using electron paramagnetic resonance, and identified the protein protons coordinating to the phosphate groups of ADP and DNA using proton-detected 31P,1H solid-state nuclear magnetic resonance spectroscopy at fast magic-angle spinning > 100 kHz, as well as temperature-dependent proton chemical-shift values to prove their engagements in hydrogen bonds. 19F and 27Al MAS NMR spectra reveal a highly mobile, fast-rotating aluminum fluoride unit pointing to the capture of a late ATP hydrolysis transition state in which the phosphoryl unit is already detached from the arginine and lysine fingers.


Subject(s)
Adenosine Diphosphate/chemistry , Adenosine Triphosphate/chemistry , Bacterial Proteins/chemistry , DNA, Bacterial/chemistry , DnaB Helicases/chemistry , Helicobacter pylori/enzymology , Adenosine Diphosphate/metabolism , Adenosine Triphosphate/metabolism , Aluminum Compounds/chemistry , Aluminum Compounds/metabolism , Arginine/chemistry , Arginine/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Catalytic Domain , Cloning, Molecular , DNA, Bacterial/genetics , DNA, Bacterial/metabolism , DnaB Helicases/genetics , DnaB Helicases/metabolism , Escherichia coli/enzymology , Escherichia coli/genetics , Fluorides/chemistry , Fluorides/metabolism , Gene Expression , Helicobacter pylori/genetics , Hydrolysis , Lysine/chemistry , Lysine/metabolism , Models, Molecular , Nuclear Magnetic Resonance, Biomolecular , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Substrate Specificity , Thermodynamics
16.
Microbiol Spectr ; 9(2): e0030121, 2021 10 31.
Article in English | MEDLINE | ID: mdl-34549994

ABSTRACT

Intervening proteins, or inteins, are mobile genetic elements that are translated within host polypeptides and removed at the protein level by splicing. In protein splicing, a self-mediated reaction removes the intein, leaving a peptide bond in place. While protein splicing can proceed in the absence of external cofactors, several examples of conditional protein splicing (CPS) have emerged. In CPS, the rate and accuracy of splicing are highly dependent on environmental conditions. Because the activity of the intein-containing host protein is compromised prior to splicing and inteins are highly abundant in the microbial world, CPS represents an emerging form of posttranslational regulation that is potentially widespread in microbes. Reactive chlorine species (RCS) are highly potent oxidants encountered by bacteria in a variety of natural environments, including within cells of the mammalian innate immune system. Here, we demonstrate that two naturally occurring RCS, namely, hypochlorous acid (the active compound in bleach) and N-chlorotaurine, can reversibly block splicing of DnaB inteins from Mycobacterium leprae and Mycobacterium smegmatis in vitro. Further, using a reporter that monitors DnaB intein activity within M. smegmatis, we show that DnaB protein splicing is inhibited by RCS in the native host. DnaB, an essential replicative helicase, is the most common intein-housing protein in bacteria. These results add to the growing list of environmental conditions that are relevant to the survival of the intein-containing host and influence protein splicing, as well as suggesting a novel mycobacterial response to RCS. We propose a model in which DnaB splicing, and therefore replication, is paused when these mycobacteria encounter RCS. IMPORTANCE Inteins are both widespread and abundant in microbes, including within several bacterial and fungal pathogens. Inteins are domains translated within host proteins and removed at the protein level by splicing. Traditionally considered molecular parasites, some inteins have emerged in recent years as adaptive posttranslational regulatory elements. Several studies have demonstrated CPS, in which the rate and accuracy of protein splicing, and thus host protein functions, are responsive to environmental conditions relevant to the intein-containing organism. In this work, we demonstrate that two naturally occurring RCS, including the active compound in household bleach, reversibly inhibit protein splicing of Mycobacterium leprae and Mycobacterium smegmatis DnaB inteins. In addition to describing a new physiologically relevant condition that can temporarily inhibit protein splicing, this study suggests a novel stress response in Mycobacterium, a bacterial genus of tremendous importance to humans.


Subject(s)
Chlorine/pharmacology , DnaB Helicases/antagonists & inhibitors , Inteins/genetics , Mycobacterium leprae/genetics , Mycobacterium smegmatis/genetics , Protein Splicing/drug effects , Chloramines/pharmacology , Chlorine/chemistry , DNA Replication/drug effects , DNA Replication/genetics , DnaB Helicases/genetics , DnaB Helicases/metabolism , Gene Expression Regulation, Bacterial/genetics , Hypochlorous Acid/pharmacology , Mycobacterium leprae/metabolism , Mycobacterium smegmatis/metabolism , Oxidants/pharmacology , Oxidation-Reduction , Protein Splicing/physiology , Reactive Oxygen Species/metabolism , Taurine/analogs & derivatives , Taurine/pharmacology
17.
Nucleic Acids Res ; 49(12): 6804-6816, 2021 07 09.
Article in English | MEDLINE | ID: mdl-34139009

ABSTRACT

In Escherichia coli, the DnaB helicase forms the basis for the assembly of the DNA replication complex. The stability of DnaB at the replication fork is likely important for successful replication initiation and progression. Single-molecule experiments have significantly changed the classical model of highly stable replication machines by showing that components exchange with free molecules from the environment. However, due to technical limitations, accurate assessments of DnaB stability in the context of replication are lacking. Using in vitro fluorescence single-molecule imaging, we visualise DnaB loaded on forked DNA templates. That these helicases are highly stable at replication forks, indicated by their observed dwell time of ∼30 min. Addition of the remaining replication factors results in a single DnaB helicase integrated as part of an active replisome. In contrast to the dynamic behaviour of other replisome components, DnaB is maintained within the replisome for the entirety of the replication process. Interestingly, we observe a transient interaction of additional helicases with the replication fork. This interaction is dependent on the τ subunit of the clamp-loader complex. Collectively, our single-molecule observations solidify the role of the DnaB helicase as the stable anchor of the replisome, but also reveal its capacity for dynamic interactions.


Subject(s)
DNA Replication , DnaB Helicases/metabolism , DNA-Directed DNA Polymerase , Escherichia coli/genetics , Multienzyme Complexes , Single Molecule Imaging
18.
Nucleic Acids Res ; 49(11): 6569-6586, 2021 06 21.
Article in English | MEDLINE | ID: mdl-34107018

ABSTRACT

Replicative helicases are essential proteins that unwind DNA in front of replication forks. Their loading depends on accessory proteins and in bacteria, DnaC and DnaI are well characterized loaders. However, most bacteria do not express either of these two proteins. Instead, they are proposed to rely on DciA, an ancestral protein unrelated to DnaC/I. While the DciA structure from Vibrio cholerae shares no homology with DnaC, it reveals similarities with DnaA and DnaX, two proteins involved during replication initiation. As other bacterial replicative helicases, VcDnaB adopts a toroid-shaped homo-hexameric structure, but with a slightly open dynamic conformation in the free state. We show that VcDnaB can load itself on DNA in vitro and that VcDciA stimulates this function, resulting in an increased DNA unwinding. VcDciA interacts with VcDnaB with a 3/6 stoichiometry and we show that a determinant residue, which discriminates DciA- and DnaC/I-helicases, is critical in vivo. Our work is the first step toward the understanding of the ancestral mode of loading of bacterial replicative helicases on DNA. It sheds light on the strategy employed by phage helicase loaders to hijack bacterial replicative helicases and may explain the recurrent domestication of dnaC/I through evolution in bacteria.


Subject(s)
Bacterial Proteins/chemistry , DNA-Binding Proteins/chemistry , DnaB Helicases/chemistry , Vibrio cholerae/enzymology , Bacterial Proteins/metabolism , DNA/metabolism , DNA-Binding Proteins/metabolism , DnaB Helicases/metabolism , Models, Molecular , Protein Conformation , Serine/chemistry
19.
Elife ; 102021 05 26.
Article in English | MEDLINE | ID: mdl-34036936

ABSTRACT

In many bacteria and eukaryotes, replication fork establishment requires the controlled loading of hexameric, ring-shaped helicases around DNA by AAA+(ATPases Associated with various cellular Activities) ATPases. How loading factors use ATP to control helicase deposition is poorly understood. Here, we dissect how specific ATPase elements of Escherichia coli DnaC, an archetypal loader for the bacterial DnaB helicase, play distinct roles in helicase loading and the activation of DNA unwinding. We have identified a new element, the arginine-coupler, which regulates the switch-like behavior of DnaC to prevent futile ATPase cycling and maintains loader responsiveness to replication restart systems. Our data help explain how the ATPase cycle of a AAA+-family helicase loader is channeled into productive action on its target; comparative studies indicate that elements analogous to the Arg-coupler are present in related, switch-like AAA+ proteins that control replicative helicase loading in eukaryotes, as well as in polymerase clamp loading and certain classes of DNA transposases.


Subject(s)
Adenosine Triphosphate/metabolism , DNA Replication , DNA, Bacterial/biosynthesis , DnaB Helicases/metabolism , Escherichia coli Proteins/metabolism , Escherichia coli/enzymology , Binding Sites , DNA, Bacterial/genetics , DnaB Helicases/genetics , Escherichia coli/genetics , Escherichia coli Proteins/genetics , Gene Expression Regulation, Bacterial , Models, Molecular , Nucleic Acid Conformation , Protein Binding , Protein Conformation , Structure-Activity Relationship
20.
Chemistry ; 27(28): 7745-7755, 2021 May 17.
Article in English | MEDLINE | ID: mdl-33822417

ABSTRACT

Paramagnetic metal ions can be inserted into ATP-fueled motor proteins by exchanging the diamagnetic Mg2+ cofactor with Mn2+ or Co2+ . Then, paramagnetic relaxation enhancement (PRE) or pseudo-contact shifts (PCSs) can be measured to report on the localization of the metal ion within the protein. We determine the metal position in the oligomeric bacterial DnaB helicase from Helicobacter pylori complexed with the transition-state ATP-analogue ADP:AlF4 - and single-stranded DNA using solid-state NMR and a structure-calculation protocol employing CYANA. We discuss and compare the use of Mn2+ and Co2+ in localizing the ATP cofactor in large oligomeric protein assemblies. 31 P PCSs induced in the Co2+ -containing sample are then used to localize the DNA phosphate groups on the Co2+ PCS tensor surface enabling structural insights into DNA binding to the DnaB helicase.


Subject(s)
DNA, Single-Stranded , Helicobacter pylori , Bacterial Proteins , DnaB Helicases/metabolism , Ions , Magnetic Resonance Spectroscopy
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