scholarly journals CRISPR-BEST: a highly efficient DSB-free base editor for filamentous actinomycetes

2019 ◽  
Author(s):  
Yaojun Tong ◽  
Helene L. Robertsen ◽  
Kai Blin ◽  
Andreas K. Klitgaard ◽  
Tilmann Weber ◽  
...  
Keyword(s):  
Genome Instability ◽  
Cytidine Deaminase ◽  
Double Strand Breaks ◽  
Antimicrobial Compounds ◽  
Dna Double Strand Breaks ◽  
Single Nucleotide ◽  
Strand Breaks ◽  
Genetic Manipulations ◽  
Nucleotide Resolution ◽  
Single Nucleotide Resolution

AbstractFilamentous actinomycetes serve as major producers of various natural products including antimicrobial compounds. Although CRISPR-Cas9 systems have been developed for more robust genetic manipulations, concerns of genome instability caused by the DNA double-strand breaks (DSB) and the toxicity of Cas9 remain. To overcome these limitations, here we report development of the DSB-free, single-nucleotide resolution genome editing system CRISPR-BEST (CRISPR-Base Editing SysTem). Specifically targeted by an sgRNA, the cytidine deaminase component of CRISPR-BEST efficiently converts C:G to T:A within a window of approximately seven-nucleotides. The system was validated and successfully used in different Streptomyces species.

scholarly journals Emerging Technologies for Genome-Wide Profiling of DNA Breakage

2021 ◽  
Vol 11 ◽  
Author(s):  
Matthew J. Rybin ◽  
Melina Ramic ◽  
Natalie R. Ricciardi ◽  
Philipp Kapranov ◽  
Claes Wahlestedt ◽  
...  
Keyword(s):  
Genome Instability ◽  
Dna Double Strand Breaks ◽  
Single Nucleotide ◽  
Strand Breaks ◽  
Single Strand Breaks ◽  
Genome Wide ◽  
A Genome ◽  
Wide Scale ◽  
Nucleotide Resolution ◽  
Genomic Regions

Genome instability is associated with myriad human diseases and is a well-known feature of both cancer and neurodegenerative disease. Until recently, the ability to assess DNA damage—the principal driver of genome instability—was limited to relatively imprecise methods or restricted to studying predefined genomic regions. Recently, new techniques for detecting DNA double strand breaks (DSBs) and single strand breaks (SSBs) with next-generation sequencing on a genome-wide scale with single nucleotide resolution have emerged. With these new tools, efforts are underway to define the “breakome” in normal aging and disease. Here, we compare the relative strengths and weaknesses of these technologies and their potential application to studying neurodegenerative diseases.

scholarly journals Locally, Meiotic Double-Strand Breaks Targeted by Gal4BD-Spo11 Occur at Discrete Sites with a Sequence Preference

2009 ◽  
Vol 29 (13) ◽  
pp. 3500-3516 ◽  
Author(s):  
Hajime Murakami ◽  
Alain Nicolas
Keyword(s):  
Hot Spots ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Single Nucleotide ◽  
Strand Breaks ◽  
Sequence Preference ◽  
Meiotic Dsbs ◽  
Resolution Mapping ◽  
Nucleotide Resolution ◽  
Recombination Hot Spots

ABSTRACT Meiotic recombination is initiated by DNA double-strand breaks (DSBs) that are catalyzed by the type II topoisomerase-like Spo11 protein. Locally, at recombination hot spots, Spo11 introduces DSBs at multiple positions within ∼75 to 250 bp, corresponding to accessible regions of the chromatin. The molecular basis of this multiplicity of cleavage positions, observed in a population of meiotic cells, remains elusive. To address this issue, we have examined the properties of the Gal4BD-Spo11 fusion protein, which targets meiotic DSBs to regions with Gal4 binding sites (UAS). By single-nucleotide resolution mapping of targeted DSBs, we found that DSB formation was restricted to discrete sites approximately 20 nucleotides from the UAS, defining a “DSB targeting window.” Thus, the multiplicity of cleavage positions at natural Spo11 hot spots likely represents binding of Spo11 to different distinct sites within the accessible DNA region in each different meiotic cell. Further, we showed that mutations in the Spo11 moiety affected the DSB distribution in the DSB targeting window and that mutations in the DNA at the Spo11 cleavage site affected DSB position. These results demonstrate that Spo11 itself has sequence preference and contributes to the choice of DSB positions.

scholarly journals Double-strand breaks are not the main cause of spontaneous sister chromatid exchange in wild-type yeast cells

2017 ◽  
Author(s):  
Clémence Claussin ◽  
David Porubský ◽  
Diana C.J. Spierings ◽  
Nancy Halsema ◽  
Stefan Rentas ◽  
...  
Keyword(s):  
Sister Chromatid ◽  
Sister Chromatid Exchange ◽  
Genome Instability ◽  
Single Cells ◽  
Double Strand Breaks ◽  
Yeast Cells ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Template Strand ◽  
Sister Chromatids

SummaryHomologous recombination involving sister chromatids is the most accurate, and thus most frequently used, form of recombination-mediated DNA repair. Despite its importance, sister chromatid recombination is not easily studied because it does not result in a change in DNA sequence, making recombination between sister chromatids difficult to detect. We have previously developed a novel DNA template strand sequencing technique, called Strand-seq, that can be used to map sister chromatid exchange (SCE) events genome-wide in single cells. An increase in the rate of SCE is an indicator of elevated recombination activity and of genome instability, which is a hallmark of cancer. In this study, we have adapted Strand-seq to detect SCE in the yeast Saccharomyces cerevisiae. Contrary to what is commonly thought, we find that most spontaneous SCE events are not due to the repair of DNA double-strand breaks.

scholarly journals Structural mechanism of DNA-end synapsis in the non-homologous end joining pathway for repairing double-strand breaks: bridge over troubled ends

2019 ◽  
Vol 47 (6) ◽  
pp. 1609-1619 ◽  
Author(s):  
Qian Wu
Keyword(s):  
Single Molecule ◽  
Genome Instability ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Structural Mechanism ◽  
Strand Breaks ◽  
Single Molecule Studies ◽  
Non Homologous End Joining ◽  
Dna Ends

Non-homologous end joining (NHEJ) is a major repair pathway for DNA double-strand breaks (DSBs), which is the most toxic DNA damage in cells. Unrepaired DSBs can cause genome instability, tumorigenesis or cell death. DNA end synapsis is the first and probably the most important step of the NHEJ pathway, aiming to bring two broken DNA ends close together and provide structural stability for end processing and ligation. This process is mediated through a group of NHEJ proteins forming higher-order complexes, to recognise and bridge two DNA ends. Spatial and temporal understanding of the structural mechanism of DNA-end synapsis has been largely advanced through recent structural and single-molecule studies of NHEJ proteins. This review focuses on core NHEJ proteins that mediate DNA end synapsis through their unique structures and interaction properties, as well as how they play roles as anchor and linker proteins during the process of ‘bridge over troubled ends'.

scholarly journals A role for nuclear envelope–bridging complexes in homology-directed repair

2014 ◽  
Vol 25 (16) ◽  
pp. 2461-2471 ◽  
Author(s):  
Rebecca K. Swartz ◽  
Elisa C. Rodriguez ◽  
Megan C. King
Keyword(s):  
Nuclear Envelope ◽  
Genome Instability ◽  
Double Strand Breaks ◽  
Linc Complex ◽  
Dna Double Strand Breaks ◽  
Cytoplasmic Microtubule ◽  
Strand Breaks ◽  
Homology Directed Repair ◽  
Cytoplasmic Microtubules ◽  
Atr Kinase

Unless efficiently and faithfully repaired, DNA double-strand breaks (DSBs) cause genome instability. We implicate a Schizosaccharomyces pombe nuclear envelope–spanning linker of nucleoskeleton and cytoskeleton (LINC) complex, composed of the Sad1/Unc84 protein Sad1 and Klarsicht/Anc1/SYNE1 homology protein Kms1, in the repair of DSBs. An induced DSB associates with Sad1 and Kms1 in S/G2 phases of the cell cycle, connecting the DSB to cytoplasmic microtubules. DSB resection to generate single-stranded DNA and the ATR kinase drive the formation of Sad1 foci in response to DNA damage. Depolymerization of microtubules or loss of Kms1 leads to an increase in the number and size of DSB-induced Sad1 foci. Further, Kms1 and the cytoplasmic microtubule regulator Mto1 promote the repair of an induced DSB by gene conversion, a type of homology-directed repair. kms1 genetically interacts with a number of genes involved in homology-directed repair; these same gene products appear to attenuate the formation or promote resolution of DSB-induced Sad1 foci. We suggest that the connection of DSBs with the cytoskeleton through the LINC complex may serve as an input to repair mechanism choice and efficiency.

scholarly journals DNA repair goes hip-hop: SMARCA and CHD chromatin remodellers join the break dance

2017 ◽  
Vol 372 (1731) ◽  
pp. 20160285 ◽  
Author(s):  
Magdalena B. Rother ◽  
Haico van Attikum
Keyword(s):  
Dna Repair ◽  
Posttranslational Modifications ◽  
Chromatin Structure ◽  
Hip Hop ◽  
Genome Stability ◽  
Genome Instability ◽  
Current Knowledge ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks

Proper signalling and repair of DNA double-strand breaks (DSB) is critical to prevent genome instability and diseases such as cancer. The packaging of DNA into chromatin, however, has evolved as a mere obstacle to these DSB responses. Posttranslational modifications and ATP-dependent chromatin remodelling help to overcome this barrier by modulating nucleosome structures and allow signalling and repair machineries access to DSBs in chromatin. Here we recap our current knowledge on how ATP-dependent SMARCA- and CHD-type chromatin remodellers alter chromatin structure during the signalling and repair of DSBs and discuss how their dysfunction impacts genome stability and human disease. This article is part of the themed issue ‘Chromatin modifiers and remodellers in DNA repair and signalling’.

scholarly journals Bridging of nucleosome-proximal DNA double-strand breaks by PARP2 enhances its interaction with HPF1

2019 ◽  
Author(s):  
Guillaume Gaullier ◽  
Genevieve Roberts ◽  
Uma M. Muthurajan ◽  
Samuel Bowerman ◽  
Johannes Rudolph ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Genome Instability ◽  
Structural Information ◽  
Regulatory Protein ◽  
Strand Break ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Damage Response

AbstractPoly(ADP-ribose) Polymerase 2 (PARP2) is one of three DNA-dependent PARPs involved in the detection of DNA damage. Upon binding to DNA double-strand breaks, PARP2 uses nicotinamide adenine dinucleotide to synthesize poly(ADP-ribose) (PAR) onto itself and other proteins, including histones. PAR chains in turn promote the DNA damage response by recruiting downstream repair factors. These early steps of DNA damage signaling are relevant for understanding how genome integrity is maintained and how their failure leads to genome instability or cancer. There is no structural information on DNA double-strand break detection in the context of chromatin. Here we present a cryo-EM structure of two nucleosomes bridged by human PARP2 and confirm that PARP2 bridges DNA ends in the context of nucleosomes bearing short linker DNA. We demonstrate that the conformation of PARP2 bound to damaged chromatin provides a binding platform for the regulatory protein Histone PARylation Factor 1 (HPF1), and that the resulting HPF1•PARP2•nucleosome complex is enzymatically active. Our results contribute to a structural view of the early steps of the DNA damage response in chromatin.

scholarly journals Identification of a signature of evolutionarily conserved stress-induced mutagenesis in cancer

2021 ◽  
Author(s):  
Luis Humberto Cisneros ◽  
Kimberly J Bussey ◽  
Charles Vasque
Keyword(s):  
Double Strand Breaks ◽  
Whole Genome Sequencing Data ◽  
Dna Double Strand Breaks ◽  
Sequencing Data ◽  
Single Nucleotide ◽  
Strand Breaks ◽  
Evolutionarily Conserved ◽  
Induced Mutagenesis ◽  
Genomic Locations ◽  
Inherited Mutations

The clustering of mutations observed in cancer cells is reminiscent of the stress-induced mutagenesis (SIM) response in bacteria. SIM employs error-prone polymerases resulting in mutations concentrated around DNA double-strand breaks with an abundance that decays with genomic distance. We performed a quantitative study on single nucleotide variant calls for whole-genome sequencing data from 1950 tumors and non-inherited mutations from 129 normal samples. We introduce statistical methods to identify mutational clusters and quantify their distribution pattern. Our results show that mutations in both normal and cancer samples are indeed clustered and have shapes indicative of SIM. We found the genomic locations of groups of close mutations are more likely to be prevalent across normal samples than in cancer suggesting loss of regulation over the mutational process during carcinogenesis.

scholarly journals Sequencing uracil in DNA at single-nucleotide resolution

2021 ◽  
Author(s):  
Liudan Jiang ◽  
Jiayong Yin ◽  
Maoxiang Qian ◽  
Shaoqin Rong ◽  
Kejing Chen ◽  
...  
Keyword(s):  
Genome Mapping ◽  
Cytidine Deaminase ◽  
Covalent Bond ◽  
Replication Timing ◽  
Cytosine Deamination ◽  
Single Nucleotide ◽  
Synthetic Dna ◽  
Mammalian Genomes ◽  
Nucleotide Resolution ◽  
Single Nucleotide Resolution

As an aberrant base in DNA, uracil is generated by dUMP misincorporation or cytosine deamination, and involved in multiple physiological and pathological processes. Current methods for whole-genome mapping of uracil all rely on uracil-DNA N-glycosylase (UNG) and are limited in resolution or specificity. Here, we present a UNG-independent Single-Nucleotide resolution Uracil Sequencing (SNU-seq) method utilizing the UdgX protein which specifically excises the uracil and forms a covalent bond with the resulting deoxyribose. SNU-seq was validated on synthetic DNA and applied to mammalian genomes. We found that the uracil content of pemetrexed-treated cells fluctuated along with DNA replication timing. We also accurately detected uracil introduced through cytosine deamination by the cytosine base editor (nCas9-APOBEC) and verified uracil occurrence in "WRC" motif within Activation-Induced Cytidine Deaminase (AID) hotspot regions in CSR-activated UNG-/- B cells.

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