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 Parp3 promotes long-range end-joining in murine cells

2018 ◽  
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 ◽  
Double Strand Breaks ◽  
Nonhomologous End Joining ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Class Switch

AbstractChromosomal 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 classical‐ and alternative-nonhomologous end joining factors (NHEJ). We reported that poly(ADP)ribose polymerase 3 (PARP3) promotes chromosomal rearrangements induced by endonucleases in multiple human cell types. In contrast to c-NHEJ factors, we show here that 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 generate Eml4-Alk fusions. In mESCs, Parp3-deficient cells had shorter deletion lengths at translocation junctions. This was corroborated using next-generation sequencing of Eml4-Alk junctions 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 Parp1 may suppress DSB 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 PARP3 is a promoter of chromosomal rearrangements and limits G4 DNA

2017 ◽  
Vol 8 (1) ◽  
Author(s):  
Tovah A. Day ◽  
Jacob V. Layer ◽  
J. Patrick Cleary ◽  
Srijoy Guha ◽  
Kristen E. Stevenson ◽  
...  
Keyword(s):  
Synthetic Lethality ◽  
Chromosomal Rearrangements ◽  
Cell Types ◽  
Nonhomologous End Joining ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Factors Affecting ◽  
Strand Breaks ◽  
G4 Dna ◽  
G Quadruplex

Abstract Chromosomal rearrangements are essential events in the pathogenesis of both malignant and nonmalignant disorders, yet the factors affecting their formation are incompletely understood. Here we develop a zinc-finger nuclease translocation reporter and screen for factors that modulate rearrangements in human cells. We identify UBC9 and RAD50 as suppressors and 53BP1, DDB1 and poly(ADP)ribose polymerase 3 (PARP3) as promoters of chromosomal rearrangements across human cell types. We focus on PARP3 as it is dispensable for murine viability and has druggable catalytic activity. We find that PARP3 regulates G quadruplex (G4) DNA in response to DNA damage, which suppresses repair by nonhomologous end-joining and homologous recombination. Chemical stabilization of G4 DNA in PARP3 −/− cells leads to widespread DNA double-strand breaks and synthetic lethality. We propose a model in which PARP3 suppresses G4 DNA and facilitates DNA repair by multiple pathways.

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 Akt: A Double-Edged Sword in Cell Proliferation and Genome Stability

2012 ◽  
Vol 2012 ◽  
pp. 1-15 ◽  
Author(s):  
Naihan Xu ◽  
Yuanzhi Lao ◽  
Yaou Zhang ◽  
David A. Gillespie
Keyword(s):  
Genome Stability ◽  
Strand Break ◽  
Nonhomologous End Joining ◽  
Dependent Manner ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Cell Behaviour ◽  
Cell Cycles ◽  
Strand Breaks ◽  
Recombination Repair

The Akt family of serine/threonine protein kinases are key regulators of multiple aspects of cell behaviour, including proliferation, survival, metabolism, and tumorigenesis. Growth-factor-activated Akt signalling promotes progression through normal, unperturbed cell cycles by acting on diverse downstream factors involved in controlling the G1/S and G2/M transitions. Remarkably, several recent studies have also implicated Akt in modulating DNA damage responses and genome stability. High Akt activity can suppress ATR/Chk1 signalling and homologous recombination repair (HRR) via direct phosphorylation of Chk1 or TopBP1 or, indirectly, by inhibiting recruitment of double-strand break (DSB) resection factors, such as RPA, Brca1, and Rad51, to sites of damage. Loss of checkpoint and/or HRR proficiency is therefore a potential cause of genomic instability in tumor cells with high Akt. Conversely, Akt is activated by DNA double-strand breaks (DSBs) in a DNA-PK- or ATM/ATR-dependent manner and in some circumstances can contribute to radioresistance by stimulating DNA repair by nonhomologous end joining (NHEJ). Akt therefore modifies both the response to and repair of genotoxic damage in complex ways that are likely to have important consequences for the therapy of tumors with deregulation of the PI3K-Akt-PTEN pathway.

scholarly journals Involvement of Artemis in nonhomologous end-joining during immunoglobulin class switch recombination

2008 ◽  
Vol 205 (13) ◽  
pp. 3031-3040 ◽  
Author(s):  
Likun Du ◽  
Mirjam van der Burg ◽  
Sergey W. Popov ◽  
Ashwin Kotnis ◽  
Jacques J.M. van Dongen ◽  
...  
Keyword(s):  
Class Switch Recombination ◽  
Double Strand Breaks ◽  
Nonhomologous End Joining ◽  
Igg Subclass ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Immunoglobulin Class ◽  
Class Switch ◽  
Human B Cells

DNA double-strand breaks (DSBs) introduced in the switch (S) regions are intermediates during immunoglobulin class switch recombination (CSR). These breaks are subsequently recognized, processed, and joined, leading to recombination of the two S regions. Nonhomologous end-joining (NHEJ) is believed to be the principle mechanism involved in DSB repair during CSR. One important component in NHEJ, Artemis, has however been considered to be dispensable for efficient CSR. In this study, we have characterized the S recombinational junctions from Artemis-deficient human B cells. Sμ–Sα junctions could be amplified from all patients tested and were characterized by a complete lack of “direct” end-joining and a remarkable shift in the use of an alternative, microhomology-based end-joining pathway. Sμ–Sγ junctions could only be amplified from one patient who carries “hypomorphic” mutations. Although these Sμ–Sγ junctions appear to be normal, a significant increase of an unusual type of sequential switching from immunoglobulin (Ig)M, through one IgG subclass, to a different IgG subclass was observed, and the Sγ–Sγ junctions showed long microhomologies. Thus, when the function of Artemis is impaired, varying modes of CSR junction resolution may be used for different S regions. Our findings strongly link Artemis to the predominant NHEJ pathway during CSR.

scholarly journals Ubiquitin-Specific-Processing Protease 47, A Novel 53BP1 regulator

2021 ◽  
Author(s):  
Doraid T. Sadideen ◽  
Baowei Chen ◽  
Manal Basili ◽  
Montaser Shaheen
Keyword(s):  
Strand Break ◽  
Dependent Manner ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Class Switch ◽  
Dna Double Strand Break ◽  
Radiation Induced ◽  
Non Homologous End Joining ◽  
Processing Protease

AbstractDNA double strand breaks (DSBs) are repair by homology-based repair or non-homologous end joining and multiple sub-pathways exist. 53BP1 is a key DNA double strand break repair protein that regulates repair pathway choice. It is key for joining DSBs during immunoglobulin heavy chain class switch recombination. Here we identify USP47 as a deubiquitylase that associates with and regulates 53BP1 function. USP47 loss results in 53BP1 instability in proteasome dependent manner, and defective 53BP1 ionizing radiation induced foci (IRIF). USP47 catalytic activity is required for maintaining 53BP1 protein level. Similar to 53BP1, USP47 depletion results in sensitivity to DNA DSB inducing agents and defective immunoglobulin CSR. Our findings establish a function for USP47 in DNA DSB repair at least partially through 53BP1.

scholarly journals 53BP1 deficiency combined with telomere dysfunction activates ATR-dependent DNA damage response

2012 ◽  
Vol 197 (2) ◽  
pp. 283-300 ◽  
Author(s):  
Paula Martínez ◽  
Juana M. Flores ◽  
Maria A. Blasco
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Ataxia Telangiectasia ◽  
Nonhomologous End Joining ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Homology Directed Repair ◽  
Damage Response

TRF1 protects mammalian telomeres from fusion and fragility. Depletion of TRF1 leads to telomere fusions as well as accumulation of γ-H2AX foci and activation of both the ataxia telangiectasia mutated (ATM)– and the ataxia telangiectasia and Rad3 related (ATR)–mediated deoxyribonucleic acid (DNA) damage response (DDR) pathways. 53BP1, which is also present at dysfunctional telomeres, is a target of ATM that accumulates at DNA double-strand breaks and favors nonhomologous end-joining (NHEJ) repair over ATM-dependent resection and homology-directed repair (homologous recombination [HR]). To address the role of 53BP1 at dysfunctional telomeres, we generated mice lacking TRF1 and 53BP1. 53BP1 deficiency significantly rescued telomere fusions in mouse embryonic fibroblasts (MEFs) lacking TRF1, but they showed evidence of a switch from the NHEJ- to HR-mediated repair of uncapped telomeres. Concomitantly, double-mutant MEFs showed evidence of hyperactivation of the ATR-dependent DDR. In intact mice, combined 53BP1/TRF1 deficiency in stratified epithelia resulted in earlier onset of DNA damage and increased CHK1 phosphorylation during embryonic development, leading to aggravation of skin phenotypes.

scholarly journals Role of the Nuclease Activity ofSaccharomyces cerevisiaeMre11 in Repair of DNA Double-Strand Breaks in Mitotic Cells

Genetics ◽  
2004 ◽  
Vol 166 (4) ◽  
pp. 1701-1713 ◽  
Author(s):  
L Kevin Lewis ◽  
Francesca Storici ◽  
Stephen Van Komen ◽  
Shanna Calero ◽  
Patrick Sung ◽  
...  
Keyword(s):  
Nuclease Activity ◽  
Double Strand Breaks ◽  
Nonhomologous End Joining ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Complex Functions ◽  
Mitotic Cells

AbstractThe Rad50:Mre11:Xrs2 (RMX) complex functions in repair of DNA double-strand breaks (DSBs) by recombination and nonhomologous end-joining (NHEJ) and is also required for telomere stability. The Mre11 subunit exhibits nuclease activities in vitro, but the role of these activities in repair in mitotic cells has not been established. In this study we have performed a comparative study of three mutants (mre11-D16A, -D56N, and -H125N) previously shown to have reduced nuclease activities in vitro. In ends-in and ends-out chromosome recombination assays using defined plasmid and oligonucleotide DNA substrates, mre11-D16A cells were as deficient as mre11 null strains, but defects were small in mre11-D56N and -H125N mutants. mre11-D16A cells, but not the other mutants, also displayed strong sensitivity to ionizing radiation, with residual resistance largely dependent on the presence of the partially redundant nuclease Exo1. mre11-D16A mutants were also most sensitive to the S-phase-dependent clastogens hydroxyurea and methyl methanesulfonate but, as previously observed for D56N and H125N mutants, were not defective in NHEJ. Importantly, the affinity of purified Mre11-D16A protein for Rad50 and Xrs2 was indistinguishable from wild type and the mutant protein formed complexes with equivalent stoichiometry. Although the role of the nuclease activity has been questioned in previous studies, the comparative data presented here suggest that the nuclease function of Mre11 is required for RMX-mediated recombinational repair and telomere stabilization in mitotic cells.

scholarly journals Role of PRKDC in cancer initiation, progression, and treatment

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Yu Chen ◽  
Yi Li ◽  
Jiani Xiong ◽  
Bin Lan ◽  
Xuefeng Wang ◽  
...  
Keyword(s):  
Checkpoint Inhibitors ◽  
Double Strand Breaks ◽  
Nonhomologous End Joining ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Cancer Initiation ◽  
Different Types ◽  
Dependent Protein

AbstractThe PRKDC gene encodes the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) protein. DNA-PKcs plays an important role in nonhomologous end joining (NHEJ) of DNA double-strand breaks (DSBs) and is also closely related to the establishment of central immune tolerance and the maintenance of chromosome stability. The occurrence and development of different types of tumors and the results of their treatment are also influenced by DNA-PKcs, and it may also predict the results of radiotherapy, chemotherapy, and therapy with immune checkpoint inhibitors (ICIs). Here, we discuss and review the structure and mechanism of action of PRKDC and DNA-PKcs and their relationship with cancer.

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