scholarly journals Genome-Wide Analysis of Cellular Response to Bacterial Genotoxin CdtB in Yeast

2007 ◽  
Vol 75 (3) ◽  
pp. 1393-1402 ◽  
Author(s):  
Takao Kitagawa ◽  
Hisashi Hoshida ◽  
Rinji Akada
Keyword(s):  
Cellular Response ◽  
Protein Translation ◽  
Strand Break ◽  
Dna Lesions ◽  
Yeast Cells ◽  
Reading Frame ◽  
Vesicular Traffic ◽  
Genome Wide ◽  
Repair Mechanisms

ABSTRACT The cytolethal distending toxins (CDTs) are secreted virulence proteins produced by several bacterial pathogens, and the subunit CdtB has the ability to create DNA lesions, primarily DNA single-strand breaks (SSBs) in vitro, and cause cell cycle arrest, cellular distension, and cell death in both mammalian and yeast cells. To elucidate the components of the mechanisms underlying the response to CdtB-induced DNA lesions, a CdtB expression plasmid was transformed into a series of diploid yeast strains harboring deletions in 4,708 nonessential genes. A total of 4,706 of these clones were successfully transformed, which we have now designated as a systematic transformation array (STA), and were subsequently screened. We identified 61 sensitive strains from the STA whose deleted genes can be categorized into a number of groups, including DNA metabolism, chromosome segregation, vesicular traffic, RNA catabolism, protein translation, morphogenesis, and nuclear transport, as well as one unknown open reading frame. However, only 28 of these strains were found to be sensitive to HO endonuclease, which is known to create a DNA double-strand break (DSB), suggesting that CdtB-induced DNA lesion is not similar to the direct DSB. Amazingly, CdtB expression elicits severe growth defects in haploid yeast cells, but only marginal defects in diploid yeast cells. The presence and absence of genes known to be involved in DNA repair in these genome-wide data reveal that CdtB-induced DNA damage is specifically repaired well in the diploid by homologous recombination but not by other repair mechanisms. Our present results provide insights into how CdtB pathogenesis is linked to eukaryotic cellular functions.

scholarly journals A Genome-Wide Proteome Array Reveals a Limited Set of Immunogens in Natural Infections of Humans and White-Footed Mice with Borrelia burgdorferi

2008 ◽  
Vol 76 (8) ◽  
pp. 3374-3389 ◽  
Author(s):  
Alan G. Barbour ◽  
Algimantas Jasinskas ◽  
Matthew A. Kayala ◽  
D. Huw Davies ◽  
Allen C. Steere ◽  
...  
Keyword(s):  
Borrelia Burgdorferi ◽  
Lyme Borreliosis ◽  
Flagellar Apparatus ◽  
Reading Frame ◽  
High Ranking ◽  
Paralogous Family ◽  
Genome Wide ◽  
A Genome ◽  
Statistical Criteria

ABSTRACT Humans and other animals with Lyme borreliosis produce antibodies to a number of components of the agent Borrelia burgdorferi, but a full accounting of the immunogens during natural infections has not been achieved. Employing a protein array produced in vitro from 1,292 DNA fragments representing ∼80% of the genome, we compared the antibody reactivities of sera from patients with early or later Lyme borreliosis to the antibody reactivities of sera from controls. Overall, ∼15% of the open reading frame (ORF) products (Orfs) of B. burgdorferi in the array detectably elicited an antibody response in humans with natural infections. Among the immunogens, 103 stood out on the basis of statistical criteria. The majority of these Orfs were also immunogenic with sera obtained from naturally infected Peromyscus leucopus mice, a major reservoir. The high-ranking set included several B. burgdorferi proteins hitherto unrecognized as immunogens, as well as several proteins that have been established as antigens. The high-ranking immunogens were more likely than nonreactive Orfs to have the following characteristics: (i) plasmid-encoded rather than chromosome-encoded proteins, (ii) a predicted lipoprotein, and (iii) a member of a paralogous family of proteins, notably the Bdr and Erp proteins. The newly discovered antigens included Orfs encoded by several ORFs of the lp36 linear plasmid, such as BBK07 and BBK19, and proteins of the flagellar apparatus, such as FliL. These results indicate that the majority of deduced proteins of B. burgdorferi do not elicit antibody responses during infection and that the limited sets of immunogens are similar for two different host species.

scholarly journals A global view of meiotic double-strand break end resection

Science ◽  
2017 ◽  
Vol 355 (6320) ◽  
pp. 40-45 ◽  
Author(s):  
Eleni P. Mimitou ◽  
Shintaro Yamada ◽  
Scott Keeney
Keyword(s):  
Meiotic Recombination ◽  
Strand Break ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Naked Dna ◽  
Global View ◽  
Genome Wide ◽  
End Resection

DNA double-strand breaks that initiate meiotic recombination are exonucleolytically processed. This 5′→3′ resection is a central, conserved feature of recombination but remains poorly understood. To address this lack, we mapped resection endpoints genome-wide at high resolution inSaccharomyces cerevisiae. Full-length resection requires Exo1 exonuclease and the DSB-responsive kinase Tel1, but not Sgs1 helicase. Tel1 also promotes efficient and timely resection initiation. Resection endpoints display pronounced heterogeneity between genomic loci that reflects a tendency for nucleosomes to block Exo1, yet Exo1 also appears to digest chromatin with high processivity and at rates similar to naked DNA in vitro. This paradox points to nucleosome destabilization or eviction as a defining feature of the meiotic resection landscape.

scholarly journals ERCC1-XPF Endonuclease Facilitates DNA Double-Strand Break Repair

2008 ◽  
Vol 28 (16) ◽  
pp. 5082-5092 ◽  
Author(s):  
Anwaar Ahmad ◽  
Andria Rasile Robinson ◽  
Anette Duensing ◽  
Ellen van Drunen ◽  
H. Berna Beverloo ◽  
...  
Keyword(s):  
Gamma Irradiation ◽  
Excision Repair ◽  
Strand Break ◽  
Dna Lesions ◽  
End Joining ◽  
Dsb Repair ◽  
Large Deletions ◽  
Mutant Cells

ABSTRACT ERCC1-XPF endonuclease is required for nucleotide excision repair (NER) of helix-distorting DNA lesions. However, mutations in ERCC1 or XPF in humans or mice cause a more severe phenotype than absence of NER, prompting a search for novel repair activities of the nuclease. In Saccharomyces cerevisiae, orthologs of ERCC1-XPF (Rad10-Rad1) participate in the repair of double-strand breaks (DSBs). Rad10-Rad1 contributes to two error-prone DSB repair pathways: microhomology-mediated end joining (a Ku86-independent mechanism) and single-strand annealing. To determine if ERCC1-XPF participates in DSB repair in mammals, mutant cells and mice were screened for sensitivity to gamma irradiation. ERCC1-XPF-deficient fibroblasts were hypersensitive to gamma irradiation, and γH2AX foci, a marker of DSBs, persisted in irradiated mutant cells, consistent with a defect in DSB repair. Mutant mice were also hypersensitive to irradiation, establishing an essential role for ERCC1-XPF in protecting against DSBs in vivo. Mice defective in both ERCC1-XPF and Ku86 were not viable. However, Ercc1 −/− Ku86 −/− fibroblasts were hypersensitive to gamma irradiation compared to single mutants and accumulated significantly greater chromosomal aberrations. Finally, in vitro repair of DSBs with 3′ overhangs led to large deletions in the absence of ERCC1-XPF. These data support the conclusion that, as in yeast, ERCC1-XPF facilitates DSB repair via an end-joining mechanism that is Ku86 independent.

scholarly journals New Faces of old Friends: Emerging new Roles of RNA-Binding Proteins in the DNA Double-Strand Break Response

2021 ◽  
Vol 8 ◽  
Author(s):  
Julie A. Klaric ◽  
Stas Wüst ◽  
Stephanie Panier
Keyword(s):  
Cellular Response ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Genome Instability ◽  
Strand Break ◽  
Dna Lesions ◽  
Cell Cycle Checkpoints ◽  
Dna Double Strand Breaks ◽  
Dna Strand

DNA double-strand breaks (DSBs) are highly cytotoxic DNA lesions. To protect genomic stability and ensure cell homeostasis, cells mount a complex signaling-based response that not only coordinates the repair of the broken DNA strand but also activates cell cycle checkpoints and, if necessary, induces cell death. The last decade has seen a flurry of studies that have identified RNA-binding proteins (RBPs) as novel regulators of the DSB response. While many of these RBPs have well-characterized roles in gene expression, it is becoming increasingly clear that they also have non-canonical functions in the DSB response that go well beyond transcription, splicing and mRNA processing. Here, we review the current understanding of how RBPs are integrated into the cellular response to DSBs and describe how these proteins directly participate in signal transduction, amplification and repair at damaged chromatin. In addition, we discuss the implications of an RBP-mediated DSB response for genome instability and age-associated diseases such as cancer and neurodegeneration.

scholarly journals The Mechanism of Replication Stalling and Recovery within Repetitive DNA

2021 ◽  
Author(s):  
Corella S Casas-Delucchi ◽  
Manuel Daza-Martin ◽  
Sophie L Williams ◽  
Gideon Coster
Keyword(s):  
Cellular Response ◽  
Repetitive Sequences ◽  
Replication Stress ◽  
Underlying Mechanism ◽  
Dna Lesions ◽  
Chromosomal Dna ◽  
Leading Strand ◽  
Fork Stalling ◽  
Dna Template

SUMMARYAccurate chromosomal DNA replication is essential to maintain genomic stability. Genetic evidence suggests that certain repetitive sequences impair replication, yet the underlying mechanism is poorly defined. Replication could be directly inhibited by the DNA template or indirectly, for example by DNA-bound proteins. Here, we reconstituted replication of mono-, di- and trinucleotide repeats in vitro using eukaryotic replisomes assembled from purified proteins. We found that structure-prone repeats are sufficient to impair replication. Whilst template unwinding was unaffected, leading strand synthesis was inhibited, leading to fork uncoupling. Synthesis through hairpin-forming repeats relied on replisome-intrinsic mechanisms, whereas synthesis of quadruplex-forming repeats required an extrinsic accessory helicase. DNA-induced fork stalling was mechanistically similar to that induced by leading strand DNA lesions, highlighting structure-prone repeats as an important potential source of replication stress. Thus, we propose that our understanding of the cellular response to replication stress also applies to stalling induced by repetitive sequences.

scholarly journals Fragment-based discovery of a new class of inhibitors targeting mycobacterial tRNA modification

2020 ◽  
Vol 48 (14) ◽  
pp. 8099-8112 ◽  
Author(s):  
Sherine E Thomas ◽  
Andrew J Whitehouse ◽  
Karen Brown ◽  
Sophie Burbaud ◽  
Juan M Belardinelli ◽  
...  
Keyword(s):  
Rapid Development ◽  
Protein Translation ◽  
Mycobacterium Abscessus ◽  
Attractive Potential ◽  
Trna Modification ◽  
Reading Frame ◽  
New Class ◽  
Translational Frameshift ◽  
New Antibiotics

Abstract Translational frameshift errors are often deleterious to the synthesis of functional proteins and could therefore be promoted therapeutically to kill bacteria. TrmD (tRNA-(N(1)G37) methyltransferase) is an essential tRNA modification enzyme in bacteria that prevents +1 errors in the reading frame during protein translation and represents an attractive potential target for the development of new antibiotics. Here, we describe the application of a structure-guided fragment-based drug discovery approach to the design of a new class of inhibitors against TrmD in Mycobacterium abscessus. Fragment library screening, followed by structure-guided chemical elaboration of hits, led to the rapid development of drug-like molecules with potent in vitro TrmD inhibitory activity. Several of these compounds exhibit activity against planktonic M. abscessus and M. tuberculosis as well as against intracellular M. abscessus and M. leprae, indicating their potential as the basis for a novel class of broad-spectrum mycobacterial drugs.

Transcriptional enhancers: from prediction to functional assessment on a genome-wide scale

Genome ◽  
2020 ◽  
pp. 1-23
Author(s):  
Ian C. Tobias ◽  
Luis E. Abatti ◽  
Sakthi D. Moorthy ◽  
Shanelle Mullany ◽  
Tiegh Taylor ◽  
...  
Keyword(s):  
High Throughput Screening ◽  
Target Genes ◽  
Regulatory Sequences ◽  
Enhancer Activity ◽  
Reading Frame ◽  
Enhancer Prediction ◽  
Genome Wide ◽  
A Genome ◽  
Enhancer Function

Enhancers are cis-regulatory sequences located distally to target genes. These sequences consolidate developmental and environmental cues to coordinate gene expression in a tissue-specific manner. Enhancer function and tissue specificity depend on the expressed set of transcription factors, which recognize binding sites and recruit cofactors that regulate local chromatin organization and gene transcription. Unlike other genomic elements, enhancers are challenging to identify because they function independently of orientation, are often distant from their promoters, have poorly defined boundaries, and display no reading frame. In addition, there are no defined genetic or epigenetic features that are unambiguously associated with enhancer activity. Over recent years there have been developments in both empirical assays and computational methods for enhancer prediction. We review genome-wide tools, CRISPR advancements, and high-throughput screening approaches that have improved our ability to both observe and manipulate enhancers in vitro at the level of primary genetic sequences, chromatin states, and spatial interactions. We also highlight contemporary animal models and their importance to enhancer validation. Together, these experimental systems and techniques complement one another and broaden our understanding of enhancer function in development, evolution, and disease.

scholarly journals Yeast Genome Maintenance by the Multifunctional PIF1 DNA Helicase Family

Genes ◽  
2020 ◽  
Vol 11 (2) ◽  
pp. 224 ◽  
Author(s):  
Julius Muellner ◽  
Kristina H. Schmidt
Keyword(s):  
Cellular Response ◽  
Replication Fork ◽  
Strand Break ◽  
Dna Helicase ◽  
Yeast Cells ◽  
Genome Integrity ◽  
Yeast Genome ◽  
Functional Evaluation ◽  
Cellular Functions ◽  
Yeast Homolog

The two PIF1 family helicases in Saccharomyces cerevisiae, Rrm3, and ScPif1, associate with thousands of sites throughout the genome where they perform overlapping and distinct roles in telomere length maintenance, replication through non-histone proteins and G4 structures, lagging strand replication, replication fork convergence, the repair of DNA double-strand break ends, and transposable element mobility. ScPif1 and its fission yeast homolog Pfh1 also localize to mitochondria where they protect mitochondrial genome integrity. In addition to yeast serving as a model system for the rapid functional evaluation of human Pif1 variants, yeast cells lacking Rrm3 have proven useful for elucidating the cellular response to replication fork pausing at endogenous sites. Here, we review the increasingly important cellular functions of the yeast PIF1 helicases in maintaining genome integrity, and highlight recent advances in our understanding of their roles in facilitating fork progression through replisome barriers, their functional interactions with DNA repair, and replication stress response pathways.

scholarly journals Microhomology-mediated end joining is the principal mediator of double-strand break repair during mitochondrial DNA lesions

2016 ◽  
Vol 27 (2) ◽  
pp. 223-235 ◽  
Author(s):  
Satish Kumar Tadi ◽  
Robin Sebastian ◽  
Sumedha Dahal ◽  
Ravi K. Babu ◽  
Bibha Choudhary ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
Dna Sequences ◽  
Nuclear Dna ◽  
Ex Vivo ◽  
Strand Break ◽  
Mitochondrial Disorders ◽  
Dna Lesions ◽  
End Joining ◽  
Dsb Repair

Mitochondrial DNA (mtDNA) deletions are associated with various mitochondrial disorders. The deletions identified in humans are flanked by short, directly repeated mitochondrial DNA sequences; however, the mechanism of such DNA rearrangements has yet to be elucidated. In contrast to nuclear DNA (nDNA), mtDNA is more exposed to oxidative damage, which may result in double-strand breaks (DSBs). Although DSB repair in nDNA is well studied, repair mechanisms in mitochondria are not characterized. In the present study, we investigate the mechanisms of DSB repair in mitochondria using in vitro and ex vivo assays. Whereas classical NHEJ (C-NHEJ) is undetectable, microhomology-mediated alternative NHEJ efficiently repairs DSBs in mitochondria. Of interest, robust microhomology-mediated end joining (MMEJ) was observed with DNA substrates bearing 5-, 8-, 10-, 13-, 16-, 19-, and 22-nt microhomology. Furthermore, MMEJ efficiency was enhanced with an increase in the length of homology. Western blotting, immunoprecipitation, and protein inhibition assays suggest the involvement of CtIP, FEN1, MRE11, and PARP1 in mitochondrial MMEJ. Knockdown studies, in conjunction with other experiments, demonstrated that DNA ligase III, but not ligase IV or ligase I, is primarily responsible for the final sealing of DSBs during mitochondrial MMEJ. These observations highlight the central role of MMEJ in maintenance of mammalian mitochondrial genome integrity and is likely relevant for deletions observed in many human mitochondrial disorders.

scholarly journals Targeted DNA methylation of neurodegenerative disease genes via homology directed repair

2019 ◽  
Author(s):  
Christopher P Cali ◽  
Daniel S Park ◽  
Edward B Lee
Keyword(s):  
Dna Methylation ◽  
Neurodegenerative Disease ◽  
Cellular Response ◽  
Regulatory Region ◽  
Strand Break ◽  
Dna Methyltransferases ◽  
Disease Genes ◽  
Fusion Construct ◽  
Homology Directed Repair

Abstract DNA methyltransferases (DNMTs) are thought to be involved in the cellular response to DNA damage, thus linking DNA repair mechanisms with DNA methylation. In this study we present Homology Assisted Repair Dependent Epigenetic eNgineering (HARDEN), a novel method of targeted DNA methylation that utilizes endogenous DNA double strand break repair pathways. This method allows for stable targeted DNA methylation through the process of homology directed repair (HDR) via an in vitro methylated exogenous repair template. We demonstrate that HARDEN can be applied to the neurodegenerative disease genes C9orf72 and APP, and methylation can be induced via HDR with both single and double stranded methylated repair templates. HARDEN allows for higher targeted DNA methylation levels than a dCas9-DNMT3a fusion protein construct at C9orf72, and genome-wide methylation analysis reveals no significant off-target methylation changes when inducing methylation via HARDEN, whereas the dCas9-DNMT3a fusion construct causes global off-target methylation. HARDEN is applied to generate a patient derived iPSC model of amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD) that recapitulates DNA methylation patterns seen in patients, demonstrating that DNA methylation of the 5′ regulatory region directly reduces C9orf72 expression and increases histone H3K9 tri-methylation levels.

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