scholarly journals Targeted chromosomal Escherichia coli: dnaB exterior surface residues regulate DNA helicase behavior to maintain genomic stability and organismal fitness

PLoS Genetics ◽  
2021 ◽  
Vol 17 (11) ◽  
pp. e1009886
Author(s):  
Megan S. Behrmann ◽  
Himasha M. Perera ◽  
Joy M. Hoang ◽  
Trisha A. Venkat ◽  
Bryan J. Visser ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Replication Fork ◽  
Dna Helicase ◽  
Exterior Surface ◽  
Genomic Stress ◽  
Dna Strands ◽  
Helicase Dnab ◽  
Organismal Fitness

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.

scholarly journals Targeted chromosomal Escherichia coli:dnaB exterior surface residues regulate DNA helicase behavior to maintain genomic stability and organismal fitness

2021 ◽  
Author(s):  
Megan S Behrmann ◽  
Himasha M Perera ◽  
Joy M Hoang ◽  
Trisha A Venkat ◽  
MICHAEL TRAKSELIS
Keyword(s):  
Conformational Changes ◽  
Genomic Stability ◽  
Dna Helicase ◽  
Exterior Surface ◽  
Genomic Stress ◽  
Dna Strands ◽  
Helicase Dnab ◽  
Enzyme Functions

Helicase regulation is vital for replisome progression, where the helicase enzyme functions to unwind duplex DNA and aids in the coordination of replication fork activities. 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 DNA damage and chromosome complexity, less stable genomes, and ultimately less viable and fit strains. Notably, while two mutations stabilized fully constricted states, they have distinct effects on genomic stability, suggesting a complex relationship between helicase regulation mechanisms and faithful, efficient DNA replication. This work explores the genomic impacts of helicase dysregulation in vivo, supporting a combined dynamic regulatory mechanism involving SEW and conformational changes and relates current mechanistic understanding to functional helicase behavior.

scholarly journals Mechanistic insights into Lhr helicase function in DNA repair

2020 ◽  
Vol 477 (16) ◽  
pp. 2935-2947
Author(s):  
Ryan J. Buckley ◽  
Kevin Kramm ◽  
Christopher D. O. Cooper ◽  
Dina Grohmann ◽  
Edward L. Bolt
Keyword(s):  
Dna Repair ◽  
Dna Replication ◽  
Single Molecule ◽  
Dna Helicase ◽  
Holliday Junctions ◽  
Single Molecule Fret ◽  
Repair Enzymes ◽  
Dna Strands

The DNA helicase Large helicase-related (Lhr) is present throughout archaea, including in the Asgard and Nanoarchaea, and has homologues in bacteria and eukaryotes. It is thought to function in DNA repair but in a context that is not known. Our data show that archaeal Lhr preferentially targets DNA replication fork structures. In a genetic assay, expression of archaeal Lhr gave a phenotype identical to the replication-coupled DNA repair enzymes Hel308 and RecQ. Purified archaeal Lhr preferentially unwound model forked DNA substrates compared with DNA duplexes, flaps and Holliday junctions, and unwound them with directionality. Single-molecule FRET measurements showed that binding of Lhr to a DNA fork causes ATP-independent distortion and base-pair melting at, or close to, the fork branchpoint. ATP-dependent directional translocation of Lhr resulted in fork DNA unwinding through the ‘parental’ DNA strands. Interaction of Lhr with replication forks in vivo and in vitro suggests that it contributes to DNA repair at stalled or broken DNA replication.

Prokaryotic DNA replication mechanisms

1987 ◽  
Vol 317 (1187) ◽  
pp. 395-420 ◽  
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Replication Fork ◽  
Kinetic Studies ◽  
Dna Helicase ◽  
Gene 32 ◽  
Dna Template ◽  
Lagging Strand

The three different prokaryotic replication systems that have been most extensively studied use the same basic components for moving a DNA replication fork, even though the individual proteins are different and lack extensive amino acid sequence homology. In the T4 bacteriophage system, the components of the DNA replication complex can be grouped into functional classes as follows: DNA polymerase (gene 43 protein), helix-destabilizing protein (gene 32 protein), polymerase accessory proteins (gene 44/62 and 45 proteins), and primosome proteins (gene 41 DNA helicase and gene 61 RNA primase). DNA synthesis in the in vitro system starts by covalent addition onto the 3'OH end at a random nick on a double-stranded DNA template and proceeds to generate a replication fork that moves at about the in vivo rate, and with approximately the in vivo base-pairing fidelity. DNA is synthesized at the fork in a continuous fashion on the leading strand and in a discontinuous fashion on the lagging strand (generating short Okazaki fragments with 5'-linked pppApCpXpYpZ pentaribonucleotide primers). Kinetic studies reveal that the DNA polymerase molecule on the lagging strand stays associated with the fork as it moves. Therefore the DNA template on the lagging strand must be folded so that the stop site for the synthesis of one Okazaki fragment is adjacent to the start site for the next such fragment, allowing the polymerase and other replication proteins on the lagging strand to recycle.

CarR, a MarR-family regulator from Corynebacterium glutamicum, modulated antibiotic and aromatic compound resistance

2019 ◽  
Vol 476 (21) ◽  
pp. 3141-3159 ◽  
Author(s):  
Meiru Si ◽  
Can Chen ◽  
Zengfan Wei ◽  
Zhijin Gong ◽  
GuiZhi Li ◽  
...  
Keyword(s):  
Stress Response ◽  
Ligand Binding ◽  
Corynebacterium Glutamicum ◽  
Conformational Changes ◽  
Cysteine Oxidation ◽  
Transcriptional Regulatory ◽  
Ligand Free ◽  
Redox Sensing

Abstract MarR (multiple antibiotic resistance regulator) proteins are a family of transcriptional regulators that is prevalent in Corynebacterium glutamicum. Understanding the physiological and biochemical function of MarR homologs in C. glutamicum has focused on cysteine oxidation-based redox-sensing and substrate metabolism-involving regulators. In this study, we characterized the stress-related ligand-binding functions of the C. glutamicum MarR-type regulator CarR (C. glutamicum antibiotic-responding regulator). We demonstrate that CarR negatively regulates the expression of the carR (ncgl2886)–uspA (ncgl2887) operon and the adjacent, oppositely oriented gene ncgl2885, encoding the hypothetical deacylase DecE. We also show that CarR directly activates transcription of the ncgl2882–ncgl2884 operon, encoding the peptidoglycan synthesis operon (PSO) located upstream of carR in the opposite orientation. The addition of stress-associated ligands such as penicillin and streptomycin induced carR, uspA, decE, and PSO expression in vivo, as well as attenuated binding of CarR to operator DNA in vitro. Importantly, stress response-induced up-regulation of carR, uspA, and PSO gene expression correlated with cell resistance to β-lactam antibiotics and aromatic compounds. Six highly conserved residues in CarR were found to strongly influence its ligand binding and transcriptional regulatory properties. Collectively, the results indicate that the ligand binding of CarR induces its dissociation from the carR–uspA promoter to derepress carR and uspA transcription. Ligand-free CarR also activates PSO expression, which in turn contributes to C. glutamicum stress resistance. The outcomes indicate that the stress response mechanism of CarR in C. glutamicum occurs via ligand-induced conformational changes to the protein, not via cysteine oxidation-based thiol modifications.

scholarly journals E3 Ubiquitin Ligase SPL2 Is a Lanthanide-Binding Protein

2021 ◽  
Vol 22 (11) ◽  
pp. 5712
Author(s):  
Michał Tracz ◽  
Ireneusz Górniak ◽  
Andrzej Szczepaniak ◽  
Wojciech Białek
Keyword(s):  
Ubiquitin Ligase ◽  
Conformational Changes ◽  
Protein Interactions ◽  
E3 Ubiquitin Ligase ◽  
Lanthanide Ions ◽  
E3 Ligases ◽  
Fluorescence Measurements ◽  
Partial Unfolding

The SPL2 protein is an E3 ubiquitin ligase of unknown function. It is one of only three types of E3 ligases found in the outer membrane of plant chloroplasts. In this study, we show that the cytosolic fragment of SPL2 binds lanthanide ions, as evidenced by fluorescence measurements and circular dichroism spectroscopy. We also report that SPL2 undergoes conformational changes upon binding of both Ca2+ and La3+, as evidenced by its partial unfolding. However, these structural rearrangements do not interfere with SPL2 enzymatic activity, as the protein retains its ability to auto-ubiquitinate in vitro. The possible applications of lanthanide-based probes to identify protein interactions in vivo are also discussed. Taken together, the results of this study reveal that the SPL2 protein contains a lanthanide-binding site, showing for the first time that at least some E3 ubiquitin ligases are also capable of binding lanthanide ions.

scholarly journals The Pif1 helicase is actively inhibited during meiotic recombination which restrains gene conversion tract length

2021 ◽  
Author(s):  
Dipti Vinayak Vernekar ◽  
Giordano Reginato ◽  
Céline Adam ◽  
Lepakshi Ranjha ◽  
Florent Dingli ◽  
...  
Keyword(s):  
Gene Conversion ◽  
Meiotic Recombination ◽  
Strand Break ◽  
Dna Helicase ◽  
Clamp Loader ◽  
Repair Synthesis ◽  
Dna Repair Synthesis ◽  
Gene Conversions

Abstract Meiotic recombination ensures proper chromosome segregation to form viable gametes and results in gene conversions events between homologs. Conversion tracts are shorter in meiosis than in mitotically dividing cells. This results at least in part from the binding of a complex, containing the Mer3 helicase and the MutLβ heterodimer, to meiotic recombination intermediates. The molecular actors inhibited by this complex are elusive. The Pif1 DNA helicase is known to stimulate DNA polymerase delta (Pol δ) -mediated DNA synthesis from D-loops, allowing long synthesis required for break-induced replication. We show that Pif1 is also recruited genome wide to meiotic DNA double-strand break (DSB) sites. We further show that Pif1, through its interaction with PCNA, is required for the long gene conversions observed in the absence of MutLβ recruitment to recombination sites. In vivo, Mer3 interacts with the PCNA clamp loader RFC, and in vitro, Mer3-MutLβ ensemble inhibits Pif1-stimulated D-loop extension by Pol δ and RFC-PCNA. Mechanistically, our results suggest that Mer3-MutLβ may compete with Pif1 for binding to RFC-PCNA. Taken together, our data show that Pif1’s activity that promotes meiotic DNA repair synthesis is restrained by the Mer3-MutLβ ensemble which in turn prevents long gene conversion tracts and possibly associated mutagenesis.

scholarly journals Furin Processing and Proteolytic Activation of Semliki Forest Virus

2003 ◽  
Vol 77 (5) ◽  
pp. 2981-2989 ◽  
Author(s):  
Xinyong Zhang ◽  
Martin Fugère ◽  
Robert Day ◽  
Margaret Kielian
Keyword(s):  
Conformational Changes ◽  
Secretory Pathway ◽  
Semliki Forest Virus ◽  
Fusion Reaction ◽  
Low Ph ◽  
Protein Fusion ◽  
Proteolytic Activation ◽  
Control Virus

ABSTRACT The alphavirus Semliki Forest virus (SFV) infects cells via a low-pH-dependent membrane fusion reaction mediated by the E1 envelope protein. Fusion is regulated by the interaction of E1 with the receptor-binding protein E2. E2 is synthesized as a precursor termed “p62,” which forms a stable heterodimer with E1 and is processed late in the secretory pathway by a cellular furin-like protease. Once processing to E2 occurs, the E1/E2 heterodimer is destabilized so that it is more readily dissociated by exposure to low pH, allowing fusion and infection. We have used FD11 cells, a furin-deficient CHO cell line, to characterize the processing of p62 and its role in the control of virus fusion and infection. p62 was not cleaved in FD11 cells and cleavage was restored in FD11 cell transfectants expressing human furin. Studies of unprocessed virus produced in FD11 cells (wt/p62) demonstrated that the p62 protein was efficiently cleaved by purified furin in vitro, without requiring prior exposure to low pH. wt/p62 virus particles were also processed during their endocytic uptake in furin-containing cells, resulting in more efficient virus infection. wt/p62 virus was compared with mutant L, in which p62 cleavage was blocked by mutation of the furin-recognition motif. wt/p62 and mutant L had similar fusion properties, requiring a much lower pH than control virus to trigger fusion and fusogenic E1 conformational changes. However, the in vivo infectivity of mutant L was more strongly inhibited than that of wt/p62, due to additional effects of the mutation on virus-cell binding.

In Vivo Retrieval of a Trapped Merci Embolectomy Device: Technical Case Report

2010 ◽  
Vol 67 (3) ◽  
pp. onsE304-onsE304 ◽  
Author(s):  
Ajeet Gordhan ◽  
John Soliman
Keyword(s):  
Vertebral Artery ◽  
Conformational Changes ◽  
Basilar Artery ◽  
Technical Note ◽  
Vessel Injury ◽  
Artery Segment ◽  
In Vitro Assessment ◽  
Artery Flow

Abstract BACKGROUND AND IMPORTANCE: This technical note describes a complication related to the use of the Merci embolectomy device not previously reported. The device can induce critical flow limitation within an accessed vessel because of a combination of vasospasm and anatomic conformational changes. Furthermore, this can limit the safe removal of the device from intracranial vasculature. We present a novel rescue technique that can be used to safely retrieve the entrapped Merci device without inciting localized vessel injury. CLINICAL PRESENTATION: A 51-year-old male with embolic occlusion of the distal basilar artery and dissection-related occlusion of the left cervical vertebral underwent mechanical thrombolysis. Flow-limiting vasospasm and/or anatomic conformational changes/ telescoping of the intracranial right vertebral artery segment was induced during deployment with subsequent entrapment of the device. Reclamation of the entrapped device was performed by initially removing the Merci microcatheter. The entrapped and fixated device was then resheathed into a 4F slip catheter within the intracranial vertebral artery. The Merci device and the slip catheter were then removed. Right vertebral and proximal basilar artery flow was reestablished after removal of the Merci device. Successful clot extraction was thereafter performed using a microsnare. CONCLUSION: In vitro assessment of the device has demonstrated its propensity to induce vasospasm. In vivo entrapment of the device has not been previously reported. Successful retrieval can be achieved if the Merci device becomes entrapped and fixated. This may be an important consideration as increased utilization of the device occurs.

scholarly journals ON THE MECHANISM OF ACTION OF BLEOMYCIN : SCISSION OF DNA STRANDS IN VITRO AND IN VIVO

1969 ◽  
Vol 22 (9) ◽  
pp. 446-448 ◽  
Author(s):  
HIDEO SUZUKI ◽  
KAZUO NAGAI ◽  
HIROSHI YAMAKI ◽  
NOBUO TANAKA ◽  
HAMAO UMEZAWA
Keyword(s):  
Mechanism Of Action ◽  
Dna Strands

A dual-action peptide-containing hydrogel targets wound infection and inflammation

2020 ◽  
Vol 12 (524) ◽  
pp. eaax6601 ◽  
Author(s):  
Manoj Puthia ◽  
Marta Butrym ◽  
Jitka Petrlova ◽  
Ann-Charlotte Strömdahl ◽  
Madelene Å. Andersson ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Inflammatory Responses ◽  
Therapeutic Potential ◽  
Nuclear Factor Κb ◽  
Reporter Mice ◽  
Dual Action ◽  
Infection And Inflammation ◽  
Molecular Patterns

There is a clinical need for improved wound treatments that prevent both infection and excessive inflammation. TCP-25, a thrombin-derived peptide, is antibacterial and scavenges pathogen-associated molecular patterns (PAMPs), such as lipopolysaccharide, thereby preventing CD14 interaction and Toll-like receptor dimerization, leading to reduced downstream immune activation. Here, we describe the development of a hydrogel formulation that was functionalized with TCP-25 to target bacteria and associated PAMP-induced inflammation. In vitro studies determined the polymer prerequisites for such TCP-25–mediated dual action, favoring the use of noncharged hydrophilic hydrogels, which enabled peptide conformational changes and LPS binding. The TCP-25–functionalized hydrogels killed Gram-positive Staphylococcus aureus and Gram-negative Pseudomonas aeruginosa bacteria in vitro, as well as in experimental mouse models of subcutaneous infection. The TCP-25 hydrogel also mediated reduction of LPS-induced local inflammatory responses, as demonstrated by analysis of local cytokine production and in vivo bioimaging using nuclear factor κB (NF-κB) reporter mice. In porcine partial thickness wound models, TCP-25 prevented infection with S. aureus and reduced concentrations of proinflammatory cytokines. Proteolytic fragmentation of TCP-25 in vitro yielded a series of bioactive TCP fragments that were identical or similar to those present in wounds in vivo. Together, the results demonstrate the therapeutic potential of TCP-25 hydrogel, a wound treatment based on the body’s peptide defense, for prevention of both bacterial infection and the accompanying inflammation.

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