Persistent DNA Damage Foci and DNA Replication with a Broken Chromosome in the African Trypanosome

ABSTRACT Damaged DNA typically imposes stringent controls on eukaryotic cell cycle progression, ensuring faithful transmission of genetic material. Some DNA breaks, and the resulting rearrangements, are advantageous, however. For example, antigenic variation in the parasitic African trypanosome, Trypanosoma brucei, relies upon homologous recombination-based rearrangements of telomeric variant surface glycoprotein (VSG) genes, triggered by breaks. Surprisingly, trypanosomes with a severed telomere continued to grow while progressively losing subtelomeric DNA, suggesting a nominal telomeric DNA damage checkpoint response. Here, we monitor the single-stranded DNA-binding protein replication protein A (RPA) in response to induced, locus-specific DNA breaks in T. brucei. RPA foci accumulated at nucleolar sites following a break within ribosomal DNA and at extranucleolar sites following a break elsewhere, including adjacent to transcribed or silent telomeric VSG genes. As in other eukaryotes, RPA foci were formed in S phase and γH2A and RAD51 damage foci were disassembled prior to mitosis. Unlike in other eukaryotes, however, and regardless of the damaged locus, RPA foci persisted through the cell cycle, and these cells continued to replicate their DNA. We conclude that a DNA break, regardless of the damaged locus, fails to trigger a stringent cell cycle checkpoint in T. brucei. This DNA damage tolerance may facilitate the generation of virulence-enhancing genetic diversity, within subtelomeric domains in particular. Stringent checkpoints may be similarly lacking in some other eukaryotic cells. IMPORTANCE Chromosome damage must be repaired to prevent the proliferation of defective cells. Alternatively, cells with damage must be eliminated. This is true of human and several other cell types but may not be the case for single-celled parasites, such as trypanosomes. African trypanosomes, which cause lethal diseases in both humans and livestock, can actually exploit chromosomal damage to activate new surface coat proteins and to evade host immune responses, for example. We monitored responses to single chromosomal breaks in trypanosomes using a DNA-binding protein that, in response to DNA damage, forms nuclear foci visible using a microscope. Surprisingly, and unlike what is seen in mammalian cells, these foci persist while cells continue to divide. We also demonstrate chromosome replication even when one chromosome is broken. These results reveal a remarkable degree of damage tolerance in trypanosomes, which may suit the lifestyle of a single-celled parasite, potentially facilitating adaptation and enhancing virulence.

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The Bacterial DNA Binding Protein MatP Involved in Linking the Nucleoid Terminal Domain to the Divisome at Midcell Interacts with Lipid Membranes

mBio ◽

10.1128/mbio.00376-19 ◽

2019 ◽

Vol 10 (3) ◽

Cited By ~ 5

Author(s):

Begoña Monterroso ◽

Silvia Zorrilla ◽

Marta Sobrinos-Sanguino ◽

Miguel Ángel Robles-Ramos ◽

Carlos Alfonso ◽

...

Keyword(s):

Dna Binding ◽

Dna Sequences ◽

Lipid Membranes ◽

Binding Protein ◽

Dna Binding Protein ◽

Content Type ◽

E Coli ◽

Daughter Cells ◽

Z Ring

ABSTRACTDivision ring formation at midcell is controlled by various mechanisms inEscherichia coli, one of them being the linkage between the chromosomal Ter macrodomain and the Z-ring mediated by MatP, a DNA binding protein that organizes this macrodomain and contributes to the prevention of premature chromosome segregation. Here we show that, during cell division, just before splitting the daughter cells, MatP seems to localize close to the cytoplasmic membrane, suggesting that this protein might interact with lipids. To test this hypothesis, we investigated MatP interaction with lipidsin vitro. We found that, when encapsulated inside vesicles and microdroplets generated by microfluidics, MatP accumulates at phospholipid bilayers and monolayers matching the lipid composition in theE. coliinner membrane. MatP binding to lipids was independently confirmed using lipid-coated microbeads and biolayer interferometry assays, which suggested that the recognition is mainly hydrophobic. Interaction of MatP with the lipid membranes also occurs in the presence of the DNA sequences specifically targeted by the protein, but there is no evidence of ternary membrane/protein/DNA complexes. We propose that the association of MatP with lipids may modulate its spatiotemporal localization and its recognition of other ligands.IMPORTANCEThe division of anE. colicell into two daughter cells with equal genomic information and similar size requires duplication and segregation of the chromosome and subsequent scission of the envelope by a protein ring, the Z-ring. MatP is a DNA binding protein that contributes both to the positioning of the Z-ring at midcell and the temporal control of nucleoid segregation. Our integratedin vivoandin vitroanalysis provides evidence that MatP can interact with lipid membranes reproducing the phospholipid mixture in theE. coliinner membrane, without concomitant recruitment of the short DNA sequences specifically targeted by MatP. This observation strongly suggests that the membrane may play a role in the regulation of the function and localization of MatP, which could be relevant for the coordination of the two fundamental processes in which this protein participates, nucleoid segregation and cell division.

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DNA-Binding Protein and the Cell Cycle in Cryptothecodinium cohnii

Differentiation ◽

10.1111/j.1432-0436.1973.tb00135.x ◽

1973 ◽

Vol 1 (6) ◽

pp. 383-391 ◽

Cited By ~ 4

Author(s):

C.K. Franker ◽

Candyce D. Prichard ◽

Carol A. Lamden

Keyword(s):

Cell Cycle ◽

Dna Binding ◽

Binding Protein ◽

Dna Binding Protein

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Sequence Elements in both the Intergenic Space and the 3′ Untranslated Region of the Crithidia fasciculata KAP3 Gene Are Required for Cell Cycle Regulation of KAP3 mRNA

Eukaryotic Cell ◽

10.1128/ec.2.4.671-677.2003 ◽

2003 ◽

Vol 2 (4) ◽

pp. 671-677 ◽

Cited By ~ 12

Author(s):

Nuraly K. Avliyakulov ◽

Jane C. Hines ◽

Dan S. Ray

Keyword(s):

Cell Cycle ◽

Dna Binding ◽

Binding Protein ◽

Large Subunit ◽

Dna Binding Protein ◽

Mrna Levels ◽

Crithidia Fasciculata ◽

Consensus Sequences ◽

Transcript Processing ◽

Cycle Dependent

ABSTRACT mRNA levels of several Crithidia fasciculata genes involved in DNA metabolism have previously been found to cycle as cells progress through the cell cycle. Octamer consensus sequences in the 5′ untranslated regions (5′ UTRs) of these transcripts were shown to be required for cycling of these mRNAs. The KAP3 gene encodes a kinetoplast histone H1-like DNA binding protein, and its mRNA levels cycle in parallel with those of the kinetoplast DNA topoisomerase (TOP2), dihydrofolate reductase-thymidylate synthase (DHFR-TS), and the large subunit of the nuclear single-stranded DNA binding protein (RPA1). KAP3 mRNA contains two octamer consensus sequences in its 3′ UTR but none in its 5′ UTR. Mutation of these octamer sequences was not sufficient to prevent cycling of a sequence-tagged KAP3 mRNA expressed from a plasmid. Mutation of an octamer sequence contained on the precursor transcript but not on the mRNA, in addition to mutation of the two octamer sequences in the 3′ UTR, was necessary to abolish cycling of the mRNA. The requirement for a sequence not present on the mature mRNA indicates that regulation of the mRNA levels by the octamer sequences occurs at or prior to splicing of the transcript. Incompletely processed RNAs containing octamer sequences were also found to accumulate during the cell cycle when the mRNA levels were lowest. These RNA species hybridize to both the KAP3 coding sequence and that of the downstream drug resistance gene, indicating a lack of processing within the intergenic region separating these genes. We propose a cell cycle-dependent interference in transcript processing mediated by octamer consensus sequences as a mechanism contributing to the cycling of such transcripts.

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Z-DNA Binding Protein Mediates Host Control of Toxoplasma gondii Infection

Infection and Immunity ◽

10.1128/iai.00511-16 ◽

2016 ◽

Vol 84 (10) ◽

pp. 3063-3070 ◽

Cited By ~ 7

Author(s):

Kelly J. Pittman ◽

Patrick W. Cervantes ◽

Laura J. Knoll

Keyword(s):

Immune Response ◽

Toxoplasma Gondii ◽

Dna Binding ◽

Chronic Infection ◽

Binding Protein ◽

Dna Binding Protein ◽

Host Immune Response ◽

Activated Macrophages ◽

Content Type ◽

Z Dna

Intrinsic toToxoplasma gondiiinfection is the parasite-induced modulation of the host immune response, which ensures establishment of a chronic lifelong infection. This manipulation of the host immune response allowsT. gondiito not only dampen the ability of the host to eliminate the parasite but also trigger parasite differentiation to the slow-growing, encysted bradyzoite form. We previously used RNA sequencing (RNA-seq) to profile the transcriptomes of mice andT. gondiiduring acute and chronic stages of infection. One of the most abundant host transcripts during acute and chronic infection was Z-DNA binding protein 1 (ZBP1). In this study, we determined that ZBP1 functions to controlT. gondiigrowth. In activated macrophages isolated from ZBP1 deletion (ZBP1−/−) mice,T. gondiihas an increased rate of replication and a decreased rate of degradation. We also identified a novel function for ZBP1 as a regulator of nitric oxide (NO) production in activated macrophages, even in the absence ofT. gondiiinfection. Upon stimulation,T. gondii-infected ZBP1−/−macrophages display increased proinflammatory cytokines compared to wild-type macrophages under the same conditions. Thesein vitrophenotypes were recapitulatedin vivo, with ZBP1−/−mice having increased susceptibility to oral challenge, higher cyst burdens during chronic infection, and elevated inflammatory cytokine responses. Taken together, these results highlight a role for ZBP1 in assisting host control ofT. gondiiinfection.

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FANCJ (BACH1) helicase forms DNA damage inducible foci with replication protein A and interacts physically and functionally with the single-stranded DNA-binding protein

Blood ◽

10.1182/blood-2006-11-057273 ◽

2007 ◽

Vol 110 (7) ◽

pp. 2390-2398 ◽

Cited By ~ 74

Author(s):

Rigu Gupta ◽

Sudha Sharma ◽

Joshua A. Sommers ◽

Mark K. Kenny ◽

Sharon B. Cantor ◽

...

Keyword(s):

Dna Damage ◽

Dna Binding ◽

Binding Protein ◽

Critical Role ◽

Protein A ◽

Replication Protein A ◽

Dna Binding Protein ◽

Replication Protein ◽

Single Stranded Dna

The BRCA1 associated C-terminal helicase (BACH1, designated FANCJ) is implicated in the chromosomal instability genetic disorder Fanconi anemia (FA) and hereditary breast cancer. A critical role of FANCJ helicase may be to restart replication as a component of downstream events that occur during the repair of DNA cross-links or double-strand breaks. We investigated the potential interaction of FANCJ with replication protein A (RPA), a single-stranded DNA-binding protein implicated in both DNA replication and repair. FANCJ and RPA were shown to coimmunoprecipitate most likely through a direct interaction of FANCJ and the RPA70 subunit. Moreover, dependent on the presence of BRCA1, FANCJ colocalizes with RPA in nuclear foci after DNA damage. Our data are consistent with a model in which FANCJ associates with RPA in a DNA damage-inducible manner and through the protein interaction RPA stimulates FANCJ helicase to better unwind duplex DNA substrates. These findings identify RPA as the first regulatory partner of FANCJ. The FANCJ-RPA interaction is likely to be important for the role of the helicase to more efficiently unwind DNA repair intermediates to maintain genomic stability.

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Cell cycle-dependent migration of the DNA-binding protein Ku80 into nucleoli

Experimental Cell Research ◽

10.1016/0014-4827(92)90433-9 ◽

1992 ◽

Vol 199 (2) ◽

pp. 262-268 ◽

Cited By ~ 19

Author(s):

Li-Lan Li ◽

Ning-Hsing Yeh

Keyword(s):

Cell Cycle ◽

Dna Binding ◽

Binding Protein ◽

Dna Binding Protein ◽

Cycle Dependent

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Cloning of a mitogen-inducible gene encoding a κB DNA-binding protein with homology to the rel oncogene and to cell-cycle motifs

Nature ◽

10.1038/348076a0 ◽

1990 ◽

Vol 348 (6296) ◽

pp. 76-80 ◽

Cited By ~ 138

Author(s):

Vincent Bours ◽

Juanita Villalobos ◽

Parris R. Burd ◽

Kathleen Kelly ◽

Ulrich Siebenlist

Keyword(s):

Cell Cycle ◽

Dna Binding ◽

Binding Protein ◽

Dna Binding Protein ◽

Gene Encoding ◽

Inducible Gene

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Mechanisms of Action of Escapin, a Bactericidal Agent in the Ink Secretion of the Sea Hare Aplysia californica: Rapid and Long-Lasting DNA Condensation and Involvement of the OxyR-Regulated Oxidative Stress Pathway

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.05874-11 ◽

2012 ◽

Vol 56 (4) ◽

pp. 1725-1734 ◽

Cited By ~ 13

Author(s):

Ko-Chun Ko ◽

Phang C. Tai ◽

Charles D. Derby

Keyword(s):

Oxidative Stress ◽

Carboxylic Acid ◽

Dna Binding ◽

Binding Protein ◽

Aplysia Californica ◽

Dna Binding Protein ◽

Bactericidal Effect ◽

Dna Condensation ◽

Maximal Effect ◽

Content Type

ABSTRACTThe marine snailAplysia californicaproduces escapin, anl-amino acid oxidase, in its defensive ink. Escapin usesl-lysine to produce diverse products called escapin intermediate products ofl-lysine (EIP-K), including α-amino-ε-caproic acid, Δ1-piperidine-2-carboxylic acid, and Δ2-piperidine-2-carboxylic acid. EIP-K and H2O2together, but neither alone, is a powerful bactericide. Here, we report bactericidal mechanisms of escapin products onEscherichia coli. We show that EIP-K and H2O2together cause rapid and long-lasting DNA condensation: 2-min treatment causes significant DNA condensation and killing, and 10-min treatment causes maximal effect, lasting at least 70 h. We isolated two mutants resistant to EIP-K plus H2O2, both having a single missense mutation in the oxidation regulatory gene,oxyR. A complementation assay showed that the mutated gene,oxyR(A233V), renders resistance to EIP-K plus H2O2, and a gene dosage effect leads to reduction of resistance for strains carrying wild-typeoxyR. Temperature stress with EIP-K does not produce the bactericidal effect, suggesting the effect is due to a specific response to oxidative stress. The null mutant for any single DNA-binding protein—Dps, H-NS, Hup, Him, or MukB—was not resistant to EIP-K plus H2O2, suggesting that no single DNA-binding protein is necessary to mediate this bactericidal effect, but allowing for the possibility that EIP-K plus H2O2could function through a combination of DNA-binding proteins. The bactericidal effect of EIP-K plus H2O2was eliminated by the ferrous ion chelator 1,10-phenanthroline, and it was reduced by the hydroxyl radical scavenger thiourea, suggesting hydroxyl radicals mediate the effects of EIP-K plus H2O2.

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