scholarly journals SAMHD1 restrains aberrant nucleotide insertions at repair junctions generated by DNA end joining

2021 ◽  
Author(s):  
Ekaterina Akimova ◽  
Franz Josef Gassner ◽  
Maria Schubert ◽  
Stefan Rebhandl ◽  
Claudia Arzt ◽  
...  
Keyword(s):  
Genome Instability ◽  
Chromosomal Rearrangements ◽  
Hek293 Cells ◽  
Dna Double Strand Breaks ◽  
Dna Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Intracellular Dntp ◽  
Samhd1 Expression ◽  
Nucleotide Insertions

Abstract Aberrant end joining of DNA double strand breaks leads to chromosomal rearrangements and to insertion of nuclear or mitochondrial DNA into breakpoints, which is commonly observed in cancer cells and constitutes a major threat to genome integrity. However, the mechanisms that are causative for these insertions are largely unknown. By monitoring end joining of different linear DNA substrates introduced into HEK293 cells, as well as by examining end joining of CRISPR/Cas9 induced DNA breaks in HEK293 and HeLa cells, we provide evidence that the dNTPase activity of SAMHD1 impedes aberrant DNA resynthesis at DNA breaks during DNA end joining. Hence, SAMHD1 expression or low intracellular dNTP levels lead to shorter repair joints and impede insertion of distant DNA regions prior end repair. Our results reveal a novel role for SAMHD1 in DNA end joining and provide new insights into how loss of SAMHD1 may contribute to genome instability and cancer development.

scholarly journals The Role of RNA in DNA Breaks, Repair and Chromosomal Rearrangements

Biomolecules ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 550
Author(s):  
Matvey Mikhailovich Murashko ◽  
Ekaterina Mikhailovna Stasevich ◽  
Anton Markovich Schwartz ◽  
Dmitriy Vladimirovich Kuprash ◽  
Aksinya Nicolaevna Uvarova ◽  
...  
Keyword(s):  
Chromosomal Rearrangements ◽  
Gene Mutations ◽  
Nonhomologous End Joining ◽  
Published Data ◽  
Dna Double Strand Breaks ◽  
Dna Breaks ◽  
End Joining ◽  
Homology Directed Repair ◽  
Chromosome Aberration Frequency

Incorrect reparation of DNA double-strand breaks (DSB) leading to chromosomal rearrangements is one of oncogenesis’s primary causes. Recently published data elucidate the key role of various types of RNA in DSB formation, recognition and repair. With growing interest in RNA biology, increasing RNAs are classified as crucial at the different stages of the main pathways of DSB repair in eukaryotic cells: nonhomologous end joining (NHEJ) and homology-directed repair (HDR). Gene mutations or variation in expression levels of such RNAs can lead to local DNA repair defects, increasing the chromosome aberration frequency. Moreover, it was demonstrated that some RNAs could stimulate long-range chromosomal rearrangements. In this review, we discuss recent evidence demonstrating the role of various RNAs in DSB formation and repair. We also consider how RNA may mediate certain chromosomal rearrangements in a sequence-specific manner.

scholarly journals Dynamics of RecA-mediated repair of replication-dependent DNA breaks

2018 ◽  
Vol 217 (7) ◽  
pp. 2299-2307 ◽  
Author(s):  
Vincent Amarh ◽  
Martin A. White ◽  
David R.F. Leach
Keyword(s):  
Temporal Dynamics ◽  
Chromosomal Rearrangements ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Dna Breaks ◽  
Chromosomal Locus ◽  
Chromosomal Replication ◽  
Strand Breaks ◽  
Sister Chromosome ◽  
Temporal Efficiency

Chromosomal replication is the major source of spontaneous DNA double-strand breaks (DSBs) in living cells. Repair of these DSBs is essential for cell viability, and accuracy of repair is critical to avoid chromosomal rearrangements. Repair of replication-dependent DSBs occurs primarily by homologous recombination with a sister chromosome. However, this reaction has never been visualized at a defined chromosomal locus, so little is known about its spatial or temporal dynamics. Repair of a replication-independent DSB generated in Escherichia coli by a rare-cutting endonuclease leads to the formation of a bundle of RecA filaments. In this study, we show that in contrast, repair of a replication-dependent DSB involves a transient RecA focus localized in the central region of the cell in which the DNA is replicated. The recombining loci remain centrally located with restricted movement before segregating with little extension to the period of postreplicative sister-chromosome cohesion. The spatial and temporal efficiency of this reaction is remarkable.

scholarly journals Parp3 promotes long-range end joining in murine cells

2018 ◽  
Vol 115 (40) ◽  
pp. 10076-10081 ◽  
Author(s):  
Jacob V. Layer ◽  
J. Patrick Cleary ◽  
Alexander J. Brown ◽  
Kristen E. Stevenson ◽  
Sara N. Morrow ◽  
...  
Keyword(s):  
Chromosomal Rearrangements ◽  
Embryonic Stem ◽  
Cell Types ◽  
Strand Break ◽  
Nonhomologous End Joining ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Class Switch

Chromosomal rearrangements, including translocations, are early and essential events in the formation of many tumors. Previous studies that defined the genetic requirements for rearrangement formation have identified differences between murine and human cells, most notably in the role of classic and alternative nonhomologous end-joining (NHEJ) factors. We reported that poly(ADP)ribose polymerase 3 (PARP3) promotes chromosomal rearrangements induced by endonucleases in multiple human cell types. We show here that in contrast to classic (c-NHEJ) factors, Parp3 also promotes rearrangements in murine cells, including translocations in murine embryonic stem cells (mESCs), class–switch recombination in primary B cells, and inversions in tail fibroblasts that generateEml4–Alkfusions. In mESCs, Parp3-deficient cells had shorter deletion lengths at translocation junctions. This was corroborated using next-generation sequencing ofEml4–Alkjunctions in tail fibroblasts and is consistent with a role for Parp3 in promoting the processing of DNA double-strand breaks. We confirmed a previous report that Parp1 also promotes rearrangement formation. In contrast with Parp3, rearrangement junctions in the absence of Parp1 had longer deletion lengths, suggesting that Parp1 may suppress double-strand break processing. Together, these data indicate that Parp3 and Parp1 promote rearrangements with distinct phenotypes.

scholarly journals Structural snapshots of human DNA polymerase μ engaged on a DNA double-strand break

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Andrea M. Kaminski ◽  
John M. Pryor ◽  
Dale A. Ramsden ◽  
Thomas A. Kunkel ◽  
Lars C. Pedersen ◽  
...  
Keyword(s):  
Chromosomal Rearrangements ◽  
Strand Break ◽  
Nonhomologous End Joining ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Template Strand ◽  
Single Strand Breaks ◽  
Dna Double Strand Break ◽  
Break Site

Abstract Genomic integrity is threatened by cytotoxic DNA double-strand breaks (DSBs), which must be resolved efficiently to prevent sequence loss, chromosomal rearrangements/translocations, or cell death. Polymerase μ (Polμ) participates in DSB repair via the nonhomologous end-joining (NHEJ) pathway, by filling small sequence gaps in broken ends to create substrates ultimately ligatable by DNA Ligase IV. Here we present structures of human Polμ engaging a DSB substrate. Synapsis is mediated solely by Polμ, facilitated by single-nucleotide homology at the break site, wherein both ends of the discontinuous template strand are stabilized by a hydrogen bonding network. The active site in the quaternary Pol μ complex is poised for catalysis and nucleotide incoporation proceeds in crystallo. These structures demonstrate that Polμ may address complementary DSB substrates during NHEJ in a manner indistinguishable from single-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 Meiotic DNA repair in the nucleolus employs a non-homologous end joining mechanism

2019 ◽  
Author(s):  
Jason Sims ◽  
Gregory P. Copenhaver ◽  
Peter Schlögelhofer
Keyword(s):  
Genome Stability ◽  
Dependent Manner ◽  
Dna Double Strand Breaks ◽  
Dna Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Meiotic Dsbs ◽  
Dna Elements ◽  
Highly Repetitive Dna ◽  
Non Homologous End Joining

AbstractRibosomal RNA genes are arranged in large arrays with hundreds of rDNA units in tandem. These highly repetitive DNA elements pose a risk to genome stability since they can undergo non-allelic exchanges. During meiosis DNA double strand breaks (DSBs) are induced as part of the regular program to generate gametes. Meiotic DSBs initiate homologous recombination (HR) which subsequently ensures genetic exchange and chromosome disjunction.In Arabidopsis thaliana we demonstrate that all 45S rDNA arrays become transcriptionally active and are recruited into the nucleolus early in meiosis. This shields the rDNA from acquiring canonical meiotic chromatin modifications, meiotic cohesin and meiosis-specific DSBs. DNA breaks within the rDNA arrays are repaired in a RAD51-independent, but LIG4-dependent manner, establishing that it is non-homologous end joining (NHEJ) that maintains rDNA integrity during meiosis. Utilizing ectopically integrated rDNA repeats we validate our findings and demonstrate that the rDNA constitutes a HR-refractory genome environment.

Persistent DNA damage signaling and DNA polymerase theta promote broken chromosome segregation

2021 ◽  
Vol 220 (12) ◽  
Author(s):  
Delisa E. Clay ◽  
Heidi S. Bretscher ◽  
Erin A. Jezuit ◽  
Korie B. Bush ◽  
Donald T. Fox
Keyword(s):  
Dna Damage ◽  
Dna Polymerase ◽  
Chromosome Segregation ◽  
Genome Instability ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Dna Damage Signaling ◽  
Alternative End Joining

Cycling cells must respond to DNA double-strand breaks (DSBs) to avoid genome instability. Missegregation of chromosomes with DSBs during mitosis results in micronuclei, aberrant structures linked to disease. How cells respond to DSBs during mitosis is incompletely understood. We previously showed that Drosophilamelanogaster papillar cells lack DSB checkpoints (as observed in many cancer cells). Here, we show that papillar cells still recruit early acting repair machinery (Mre11 and RPA3) and the Fanconi anemia (FA) protein Fancd2 to DSBs. These proteins persist as foci on DSBs as cells enter mitosis. Repair foci are resolved in a stepwise manner during mitosis. DSB repair kinetics depends on both monoubiquitination of Fancd2 and the alternative end-joining protein DNA polymerase θ. Disruption of either or both of these factors causes micronuclei after DNA damage, which disrupts intestinal organogenesis. This study reveals a mechanism for how cells with inactive DSB checkpoints can respond to DNA damage that persists into mitosis.

scholarly journals Frequency of DNA end joining in trans is not determined by the predamage spatial proximity of double-strand breaks in yeast

2019 ◽  
Vol 116 (19) ◽  
pp. 9481-9490 ◽  
Author(s):  
Sham Sunder ◽  
Thomas E. Wilson
Keyword(s):  
Chromosomal Rearrangements ◽  
Double Strand Breaks ◽  
Yeast Cells ◽  
Spatial Distance ◽  
Spatial Proximity ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Repair Efficiency ◽  
In Trans

DNA double-strand breaks (DSBs) are serious genomic insults that can lead to chromosomal rearrangements if repaired incorrectly. To gain insight into the nuclear mechanisms contributing to these rearrangements, we developed an assay in yeast to measure cis (same site) vs. trans (different site) repair for the majority process of precise nonhomologous end joining (NHEJ). In the assay, the HO endonuclease gene is placed between two HO cut sites such that HO expression is self-terminated upon induction. We further placed an additional cut site in various genomic loci such that NHEJ in trans led to expression of a LEU2 reporter gene. Consistent with prior reports, cis NHEJ was more efficient than trans NHEJ. However, unlike homologous recombination, where spatial distance between a single DSB and donor locus was previously shown to correlate with repair efficiency, trans NHEJ frequency remained essentially constant regardless of the position of the two DSB loci, even when they were on the same chromosome or when two trans repair events were put in competition. Repair of similar DSBs via single-strand annealing of short terminal direct repeats showed substantially higher repair efficiency and trans repair frequency, but still without a strong correlation of trans repair to genomic position. Our results support a model in which yeast cells mobilize, and perhaps compartmentalize, multiple DSBs in a manner that no longer reflects the predamage position of two broken loci.

scholarly journals Small tandem DNA duplications result from CST-guided Pol α-primase action at DNA break termini

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Joost Schimmel ◽  
Núria Muñoz-Subirana ◽  
Hanneke Kool ◽  
Robin van Schendel ◽  
Marcel Tijsterman
Keyword(s):  
Mechanistic Explanation ◽  
Underlying Mechanism ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Dna Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Dna Break ◽  
Non Homologous End Joining ◽  
Tandem Duplications

AbstractSmall tandem duplications of DNA occur frequently in the human genome and are implicated in the aetiology of certain human cancers. Recent studies have suggested that DNA double-strand breaks are causal to this mutational class, but the underlying mechanism remains elusive. Here, we identify a crucial role for DNA polymerase α (Pol α)-primase in tandem duplication formation at breaks having complementary 3′ ssDNA protrusions. By including so-called primase deserts in CRISPR/Cas9-induced DNA break configurations, we reveal that fill-in synthesis preferentially starts at the 3′ tip, and find this activity to be dependent on 53BP1, and the CTC1-STN1-TEN1 (CST) and Shieldin complexes. This axis generates near-blunt ends specifically at DNA breaks with 3′ overhangs, which are subsequently repaired by non-homologous end-joining. Our study provides a mechanistic explanation for a mutational signature abundantly observed in the genomes of species and cancer cells.

scholarly journals Homology Directed Repair by Cas9:Donor Co-localization in Mammalian Cells

2018 ◽  
Author(s):  
Philip J.R. Roche ◽  
Heidi Gytz ◽  
Faiz Hussain ◽  
Christopher J.F. Cameron ◽  
Denis Paquette ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Gene Editing ◽  
Low Frequency ◽  
Cost Effective ◽  
Hek293 Cells ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Homology Directed Repair ◽  
Non Homologous End Joining

AbstractHomology directed repair (HDR) induced by site specific DNA double strand breaks (DSB) with CRISPR/Cas9 is a precision gene editing approach that occurs at low frequency in comparison to indel forming non homologous end joining (NHEJ). In order to obtain high HDR percentages in mammalian cells, we engineered Cas9 protein fused to a high-affinity monoavidin domain to deliver biotinylated donor DNA to a DSB site. In addition, we used the cationic polymer, polyethylenimine, to deliver Cas9 RNP-donor DNA complex into the cell. Combining these strategies improved HDR percentages of up to 90% in three tested loci (CXCR4, EMX1, and TLR) in standard HEK293 cells. Our approach offers a cost effective, simple and broadly applicable gene editing method, thereby expanding the CRISPR/Cas9 genome editing toolbox.SummaryPrecision gene editing occurs at a low percentage in mammalian cells using Cas9. Colocalization of donor with Cas9MAV and PEI delivery raises HDR occurrence.

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