scholarly journals One, No One, and One Hundred Thousand: The Many Forms of Ribonucleotides in DNA

2020 ◽  
Vol 21 (5) ◽  
pp. 1706 ◽  
Author(s):  
Giulia Maria Nava ◽  
Lavinia Grasso ◽  
Sarah Sertic ◽  
Achille Pellicioli ◽  
Marco Muzi Falconi ◽  
...  
Keyword(s):  
Genome Stability ◽  
Dna Polymerases ◽  
Dark Side ◽  
Strand Breaks ◽  
Rna:Dna Hybrids ◽  
The Many ◽  
Nucleic Acid Structures ◽  
Template Dna

In the last decade, it has become evident that RNA is frequently found in DNA. It is now well established that single embedded ribonucleoside monophosphates (rNMPs) are primarily introduced by DNA polymerases and that longer stretches of RNA can anneal to DNA, generating RNA:DNA hybrids. Among them, the most studied are R-loops, peculiar three-stranded nucleic acid structures formed upon the re-hybridization of a transcript to its template DNA. In addition, polyribonucleotide chains are synthesized to allow DNA replication priming, double-strand breaks repair, and may as well result from the direct incorporation of consecutive rNMPs by DNA polymerases. The bright side of RNA into DNA is that it contributes to regulating different physiological functions. The dark side, however, is that persistent RNA compromises genome integrity and genome stability. For these reasons, the characterization of all these structures has been under growing investigation. In this review, we discussed the origin of single and multiple ribonucleotides in the genome and in the DNA of organelles, focusing on situations where the aberrant processing of RNA:DNA hybrids may result in multiple rNMPs embedded in DNA. We concluded by providing an overview of the currently available strategies to study the presence of single and multiple ribonucleotides in DNA in vivo.

scholarly journals In vivo analysis of FANCD2 recruitment at meiotic DNA breaks in Caenorhabditis elegans

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Marcello Germoglio ◽  
Anna Valenti ◽  
Ines Gallo ◽  
Chiara Forenza ◽  
Pamela Santonicola ◽  
...  
Keyword(s):  
Dna Repair ◽  
Caenorhabditis Elegans ◽  
Genome Stability ◽  
Genetic Interaction ◽  
Model Systems ◽  
Dna Breaks ◽  
Strand Breaks ◽  
C Elegans ◽  
In Vivo Analysis

AbstractFanconi Anemia is a rare genetic disease associated with DNA repair defects, congenital abnormalities and infertility. Most of FA pathway is evolutionary conserved, allowing dissection and mechanistic studies in simpler model systems such as Caenorhabditis elegans. In the present study, we employed C. elegans to better understand the role of FA group D2 (FANCD2) protein in vivo, a key player in promoting genome stability. We report that localization of FCD-2/FANCD2 is dynamic during meiotic prophase I and requires its heterodimeric partner FNCI-1/FANCI. Strikingly, we found that FCD-2 recruitment depends on SPO-11-induced double-strand breaks (DSBs) but not RAD-51-mediated strand invasion. Furthermore, exposure to DNA damage-inducing agents boosts FCD-2 recruitment on the chromatin. Finally, analysis of genetic interaction between FCD-2 and BRC-1 (the C. elegans orthologue of mammalian BRCA1) supports a role for these proteins in different DSB repair pathways. Collectively, we showed a direct involvement of FCD-2 at DSBs and speculate on its function in driving meiotic DNA repair.

scholarly journals Unexpected behavior of DNA polymerase Mu opposite template 8-oxo-7,8-dihydro-2′-guanosine

2019 ◽  
Vol 47 (17) ◽  
pp. 9410-9422 ◽  
Author(s):  
Andrea M Kaminski ◽  
Kishore K Chiruvella ◽  
Dale A Ramsden ◽  
Thomas A Kunkel ◽  
Katarzyna Bebenek ◽  
...  
Keyword(s):  
Double Strand Breaks ◽  
Nonhomologous End Joining ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Steady State Kinetics ◽  
Ligase Iv ◽  
Template Dna ◽  
Dna Substrate

Abstract DNA double-strand breaks (DSBs) resulting from reactive oxygen species generated by exposure to UV and ionizing radiation are characterized by clusters of lesions near break sites. Such complex DSBs are repaired slowly, and their persistence can have severe consequences for human health. We have therefore probed DNA break repair containing a template 8-oxo-7,8-dihydro-2′-guanosine (8OG) by Family X Polymerase μ (Pol μ) in steady-state kinetics and cell-based assays. Pol μ tolerates 8OG-containing template DNA substrates, and the filled products can be subsequently ligated by DNA Ligase IV during Nonhomologous end-joining. Furthermore, Pol μ exhibits a strong preference for mutagenic bypass of 8OG by insertion of adenine. Crystal structures reveal that the template 8OG is accommodated in the Pol μ active site with none of the DNA substrate distortions observed for Family X siblings Pols β or λ. Kinetic characterization of template 8OG bypass indicates that Pol μ inserts adenosine nucleotides with weak sugar selectivity and, given the high cellular concentration of ATP, likely performs its role in repair of complex 8OG-containing DSBs using ribonucleotides.

scholarly journals A nucleotide resolution map of Top2-linked DNA breaks in the yeast and human genome

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
William H. Gittens ◽  
Dominic J. Johnson ◽  
Rachal M. Allison ◽  
Tim J. Cooper ◽  
Holly Thomas ◽  
...  
Keyword(s):  
Dna Cleavage ◽  
Genome Stability ◽  
Dna Double Strand Breaks ◽  
Dna Breaks ◽  
Strand Breaks ◽  
Single Strand Breaks ◽  
Human Genomes ◽  
Nucleotide Resolution

Abstract DNA topoisomerases are required to resolve DNA topological stress. Despite this essential role, abortive topoisomerase activity generates aberrant protein-linked DNA breaks, jeopardising genome stability. Here, to understand the genomic distribution and mechanisms underpinning topoisomerase-induced DNA breaks, we map Top2 DNA cleavage with strand-specific nucleotide resolution across the S. cerevisiae and human genomes—and use the meiotic Spo11 protein to validate the broad applicability of this method to explore the role of diverse topoisomerase family members. Our data characterises Mre11-dependent repair in yeast and defines two strikingly different fractions of Top2 activity in humans: tightly localised CTCF-proximal, and broadly distributed transcription-proximal, the latter correlated with gene length and expression. Moreover, single nucleotide accuracy reveals the influence primary DNA sequence has upon Top2 cleavage—distinguishing sites likely to form canonical DNA double-strand breaks (DSBs) from those predisposed to form strand-biased DNA single-strand breaks (SSBs) induced by etoposide (VP16) in vivo.

scholarly journals DNA-dependent macromolecular condensation drives self-assembly of the meiotic DNA break machinery

2020 ◽  
Author(s):  
Corentin Claeys Bouuaert ◽  
Stephen Pu ◽  
Juncheng Wang ◽  
Dinshaw J. Patel ◽  
Scott Keeney
Keyword(s):  
Self Assembly ◽  
Chromosome Structure ◽  
Coiled Coil ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Dna Breakage ◽  
And Control

Formation of meiotic DNA double-strand breaks (DSBs) by Spo11 is tightly regulated and tied to chromosome structure, but the higher-order assemblies that execute and control DNA breakage are poorly understood. We address this question through molecular characterization of Saccharomyces cerevisiae RMM proteins (Rec114, Mei4 and Mer2)—essential, conserved components of the DSB machinery. Each subcomplex of Rec114–Mei4 (2:1 heterotrimer) or Mer2 (homotetrameric coiled coil) is monodisperse in solution, but they independently condense with DNA into dynamic, reversible nucleoprotein clusters that share properties with phase-separated systems. Multivalent interactions drive condensation, which correlates with DSB formation in vivo. Condensates fuse into mixed Rec114–Mei4–Mer2 clusters that further recruit Spo11 complexes. Our data show how the DSB machinery self-assembles on chromosome axes to create centers of DSB activity. We propose that multilayered control of Spo11 arises from recruitment of regulatory components and modulation of biophysical properties of the condensates.

scholarly journals Identification and characterization of FUS/TLS as a new target of ATM

2008 ◽  
Vol 415 (2) ◽  
pp. 297-307 ◽  
Author(s):  
Mary Gardiner ◽  
Rachel Toth ◽  
Franck Vandermoere ◽  
Nicholas A. Morrice ◽  
John Rouse
Keyword(s):  
Genome Stability ◽  
Chromosomal Translocation ◽  
Strand Breaks ◽  
Homologous Protein ◽  
Fused In Sarcoma ◽  
Enhancer Binding Protein ◽  
Dependent Protein ◽  
Phosphoinositide 3 Kinase

ATM (ataxia-telangiectasia mutated), ATR (ATM- and Rad3-related) and DNA-PK (DNA-dependent protein kinase), important regulators of genome stability, belong to the PIKK (phosphoinositide 3-kinase-like kinase) family of protein kinases. In the present study, DNA-affinity chromatography was used to identify DNA-binding proteins phosphorylated by these kinases. This resulted in the identification of FUS (fused in sarcoma)/TLS (translocated in liposarcoma) as an in vitro target of the PIKKs. FUS is a member of the Ewing's sarcoma family of proteins that appears to play a role in regulating genome stability, since mice lacking FUS show chromosomal instability and defects in meiosis. The residues in FUS that are phosphorylated in vitro and in vivo were identified, and phospho-specific antibodies were generated to demonstrate that FUS becomes phosphorylated at Ser42in vivo, primarily in response to agents that cause DSBs (double-strand breaks). DSB-induced FUS phosphorylation in vivo at Ser42 requires ATM and not DNA-PK. Although Ser42 is retained in the oncogenic FUS–CHOP [C/EBP (CCAAT/enhancer-binding protein)-homologous protein 10] fusion generated by a t(12;16)(q13;p11) chromosomal translocation, Ser42 in FUS–CHOP is not phosphorylated after DNA damage. These results identify FUS as a new target of the ATM-signalling pathway and strengthen the notion that FUS regulates genome stability.

scholarly journals A nucleotide resolution map of Top2-linked DNA breaks in the yeast and human genome

2019 ◽  
Author(s):  
William Gittens ◽  
Dominic J. Johnson ◽  
Rachal M. Allison ◽  
Tim J. Cooper ◽  
Holly Thomas ◽  
...  
Keyword(s):  
Dna Cleavage ◽  
Genome Stability ◽  
Dna Double Strand Breaks ◽  
Dna Breaks ◽  
Strand Breaks ◽  
Single Strand Breaks ◽  
Human Genomes ◽  
Nucleotide Resolution

AbstractDNA topoisomerases are required to resolve DNA topological stress. Despite this essential role, abortive topoisomerase activity generates aberrant protein-linked DNA breaks, jeopardising genome stability. Here, to understand the genomic distribution and mechanisms underpinning topoisomerase-induced DNA breaks, we map Top2 DNA cleavage with strand-specific nucleotide resolution across the S. cerevisiae and human genomes—and use the meiotic Spo11 protein to validate the broad applicability of this method to explore the role of diverse topoisomerase family members. Our data characterises Mre11-dependent repair in yeast, and defines two strikingly different fractions of Top2 activity in humans: tightly localised CTCF-proximal, and broadly distributed transcription-proximal, the latter correlated with gene length and expression. Moreover, single nucleotide accuracy enables us to reveal the influence primary DNA sequence has upon Top2 cleavage—distinguishing canonical DNA double-strand breaks (DSBs) from a major population of DNA single-strand breaks (SSBs) induced by etoposide (VP16) in vivo.

scholarly journals Characterization of four virulent Klebsiella pneumoniae bacteriophages, and evaluation of their potential use in complex phage preparation

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Fedor Zurabov ◽  
Evgeniy Zhilenkov
Keyword(s):  
Host Range ◽  
Nosocomial Infections ◽  
Burst Size ◽  
In Vivo Studies ◽  
Bacterial Strains ◽  
Phage Preparation ◽  
Complex Preparation ◽  
The Many

Abstract Background Nowadays, hundreds of thousands of deaths per year are caused by antibiotic resistant nosocomial infections and the prognosis for future years is much worse, as evidenced by modern research. Bacteria of the Klebsiella genus are one of the main pathogens that cause nosocomial infections. Among the many antimicrobials offered to replace or supplement traditional antibiotics, bacteriophages are promising candidates. Methods This article presents microbiological, physicochemical and genomic characterization of 4 virulent bacteriophages belonging to Siphoviridae, Myoviridae and Podoviridae families. Phages were studied by electron microscopy; their host range, lytic activity, adsorption rate, burst size, latent period, frequency of phage-resistant forms generation, lysis dynamics and sensitivity of phage particles to temperature and pH were identified; genomes of all 4 bacteriophages were studied by restriction digestion and complete genome sequence. Results Studied phages showed wide host range and high stability at different temperature and pH values. In contrast with single phages, a cocktail of bacteriophages lysed all studied bacterial strains, moreover, no cases of the emergence of phage-resistant bacterial colonies were detected. Genomic data proved that isolated viruses do not carry antibiotic resistance, virulence or lysogenic genes. Three out of four bacteriophages encode polysaccharide depolymerases, which are involved in the degradation of biofilms and capsules. Conclusions The bacteriophages studied in this work are promising for further in vivo studies and might be used in phage therapy as part of a complex therapeutic and prophylactic phage preparation. The conducted studies showed that the complex preparation is more effective than individual phages. The use of the complex phage cocktail allows to extend the lytic spectrum, and significantly reduces the possibility of phage-resistant forms generation.

scholarly journals Unbiased detection of CRISPR off-targets in vivo using DISCOVER-Seq

2018 ◽  
Author(s):  
Beeke Wienert ◽  
Stacia K Wyman ◽  
Christopher D Richardson ◽  
Charles D Yeh ◽  
Pinar Akcakaya ◽  
...  
Keyword(s):  
Gene Editing ◽  
Double Strand Breaks ◽  
Therapeutic Gene ◽  
Strand Breaks ◽  
Cellular Models ◽  
Genome Wide ◽  
Target Sites ◽  
Single Base Resolution

AbstractGenome editing using nucleases such as CRISPR-Cas induces programmable DNA damage at a target genomic site but can also affect off-target sites. Here, we develop a powerful, sensitive assay for the unbiased identification of off-target sites that we term DISCOVER-Seq. This approach takes advantage of the recruitment of endogenous DNA repair factors for genome-wide identification of Cas-induced double-strand breaks. One such factor, MRE11, is recruited precisely to double-strand breaks, enabling molecular characterization of nuclease cut sites with single-base resolution. DISCOVER-Seq detects off-targets in cellular models and in vivo upon adenoviral gene editing of mouse livers, paving the way for real-time off-target discovery during therapeutic gene editing. DISCOVER-Seq is furthermore applicable to multiple types of Cas nucleases and provides an unprecedented view of events that precede repair of the affected sites.

scholarly journals Tβ4–Ac-SDKP pathway: Any relevance for the cardiovascular system?

2019 ◽  
Vol 97 (7) ◽  
pp. 589-599
Author(s):  
Kamal M. Kassem ◽  
Sonal Vaid ◽  
Hongmei Peng ◽  
Sarah Sarkar ◽  
Nour-Eddine Rhaleb
Keyword(s):  
Biological Effects ◽  
Endothelial Cell Migration ◽  
Renal Diseases ◽  
Putative Receptor ◽  
Therapeutic Tools ◽  
The Many ◽  
Experimental Findings

The last 20 years witnessed the emergence of the thymosin β4 (Tβ4)–N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) pathway as a new source of future therapeutic tools to treat cardiovascular and renal diseases. In this review article, we attempted to shed light on the numerous experimental findings pertaining to the many promising cardiovascular therapeutic avenues for Tβ4 and (or) its N-terminal derivative, Ac-SDKP. Specifically, Ac-SDKP is endogenously produced from the 43-amino acid Tβ4 by 2 successive enzymes, meprin α and prolyl oligopeptidase. We also discussed the possible mechanisms involved in the Tβ4–Ac-SDKP-associated cardiovascular biological effects. In infarcted myocardium, Tβ4 and Ac-SDKP facilitate cardiac repair after infarction by promoting endothelial cell migration and myocyte survival. Additionally, Tβ4 and Ac-SDKP have antifibrotic and anti-inflammatory properties in the arteries, heart, lungs, and kidneys, and stimulate both in vitro and in vivo angiogenesis. The effects of Tβ4 can be mediated directly through a putative receptor (Ku80) or via its enzymatically released N-terminal derivative Ac-SDKP. Despite the localization and characterization of Ac-SDKP binding sites in myocardium, more studies are needed to fully identify and clone Ac-SDKP receptors. It remains promising that Ac-SDKP or its degradation-resistant analogs could serve as new therapeutic tools to treat cardiac, vascular, and renal injury and dysfunction to be used alone or in combination with the already established pharmacotherapy for cardiovascular diseases.

Histone marks: repairing DNA breaks within the context of chromatin

2012 ◽  
Vol 40 (2) ◽  
pp. 370-376 ◽  
Author(s):  
Kyle M. Miller ◽  
Stephen P. Jackson
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Genome Stability ◽  
Nuclear Dna ◽  
Dna Lesions ◽  
Dna Breaks ◽  
Strand Breaks ◽  
Dna Dsbs

Inherited or acquired defects in detecting, signalling or repairing DNA damage are associated with various human pathologies, including immunodeficiencies, neurodegenerative diseases and various forms of cancer. Nuclear DNA is packaged into chromatin and therefore the true in vivo substrate of damaged DNA occurs within the context of chromatin. Our work aims to decipher the mechanisms by which cells detect DNA damage and signal its presence to the DNA-repair and cell-cycle machineries. In particular, much of our work has focused on DNA DSBs (double-strand breaks) that are generated by ionizing radiation and radiomimetic chemicals, and which can also arise when the DNA replication apparatus encounters other DNA lesions. In the present review, we describe some of our recent work, as well as the work of other laboratories, that has identified new chromatin proteins that mediate DSB responses, control SDB processing or modulate chromatin structure at DNA-damage sites. We also aim to survey several recent advances in the field that have contributed to our understanding of how particular histone modifications and involved in DNA repair. It is our hope that by understanding the role of chromatin and its modifications in promoting DNA repair and genome stability, this knowledge will provide opportunities for developing novel classes of drugs to treat human diseases, including cancer.

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