scholarly journals WRN helicase safeguards deprotected replication forks in BRCA2-mutated cancer cells

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Arindam Datta ◽  
Kajal Biswas ◽  
Joshua A. Sommers ◽  
Haley Thompson ◽  
Sanket Awate ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Genome Stability ◽  
Parp Inhibitor ◽  
Nuclease Activity ◽  
End Joining ◽  
Strand Breaks ◽  
Fork Stalling ◽  
Non Homologous End Joining ◽  
Wrn Helicase

AbstractThe tumor suppressor BRCA2 protects stalled forks from degradation to maintain genome stability. However, the molecular mechanism(s) whereby unprotected forks are stabilized remains to be fully characterized. Here, we demonstrate that WRN helicase ensures efficient restart and limits excessive degradation of stalled forks in BRCA2-deficient cancer cells. In vitro, WRN ATPase/helicase catalyzes fork restoration and curtails MRE11 nuclease activity on regressed forks. We show that WRN helicase inhibitor traps WRN on chromatin leading to rapid fork stalling and nucleolytic degradation of unprotected forks by MRE11, resulting in MUS81-dependent double-strand breaks, elevated non-homologous end-joining and chromosomal instability. WRN helicase inhibition reduces viability of BRCA2-deficient cells and potentiates cytotoxicity of a poly (ADP)ribose polymerase (PARP) inhibitor. Furthermore, BRCA2-deficient xenograft tumors in mice exhibited increased DNA damage and growth inhibition when treated with WRN helicase inhibitor. This work provides mechanistic insight into stalled fork stabilization by WRN helicase when BRCA2 is deficient.

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 To Join or Not to Join: Decision Points Along the Pathway to Double-Strand Break Repair vs. Chromosome End Protection

2021 ◽  
Vol 9 ◽  
Author(s):  
Stephanie M. Ackerson ◽  
Carlan Romney ◽  
P. Logan Schuck ◽  
Jason A. Stewart
Keyword(s):  
Genome Stability ◽  
Strand Break ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Dsb Repair ◽  
Strand Breaks ◽  
Decision Points ◽  
Chromosome Fusions ◽  
Non Homologous End Joining ◽  
Dna Ends

The regulation of DNA double-strand breaks (DSBs) and telomeres are diametrically opposed in the cell. DSBs are considered one of the most deleterious forms of DNA damage and must be quickly recognized and repaired. Telomeres, on the other hand, are specialized, stable DNA ends that must be protected from recognition as DSBs to inhibit unwanted chromosome fusions. Decisions to join DNA ends, or not, are therefore critical to genome stability. Yet, the processing of telomeres and DSBs share many commonalities. Accordingly, key decision points are used to shift DNA ends toward DSB repair vs. end protection. Additionally, DSBs can be repaired by two major pathways, namely homologous recombination (HR) and non-homologous end joining (NHEJ). The choice of which repair pathway is employed is also dictated by a series of decision points that shift the break toward HR or NHEJ. In this review, we will focus on these decision points and the mechanisms that dictate end protection vs. DSB repair and DSB repair choice.

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.

scholarly journals Evolutionary and Comparative analysis of bacterial Non-Homologous End Joining Repair

2019 ◽  
Author(s):  
Mohak Sharda ◽  
Anjana Badrinarayanan ◽  
Aswin Sai Narain Seshasayee
Keyword(s):  
Growth Rates ◽  
Genome Stability ◽  
Ancestral Reconstruction ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Domains Of Life ◽  
History Of ◽  
Non Homologous End Joining ◽  
Two Kingdoms

AbstractDNA double-strand breaks (DSBs) are a threat to genome stability. In all domains of life, DSBs are faithfully fixed via homologous recombination. Recombination requires the presence of an uncut copy of duplex DNA that is used as a template for repair. Alternatively, in the absence of a template, cells utilize error-prone Non-homologous end joining (NHEJ). Although ubiquitously found in eukaryotes, NHEJ is not universally present in bacteria. It is unclear as to why many prokaryotes lack this pathway. To understand what could have led to the current distribution of bacterial NHEJ, we carried out comparative genomics and phylogenetic analysis across ~6000 genomes. Our results show that this pathway is sporadically distributed across the phylogeny. Ancestral reconstruction further suggests that NHEJ was absent in the eubacterial ancestor, and can be acquired via specific routes. Integrating NHEJ occurrence data for archaea, we also find evidence for extensive horizontal exchange of NHEJ genes between the two kingdoms as well as across bacterial clades. The pattern of occurrence in bacteria is consistent with correlated evolution of NHEJ with key genome characteristics of genome size and growth rates; NHEJ presence is associated with large genome sizes and/or slow growth rates, with the former being the dominant correlate. Given the central role these traits play in determining the ability to carry out recombination, it is possible that the evolutionary history of bacterial NHEJ may have been shaped by requirement for efficient DSB repair.

scholarly journals Polθ inhibitors elicit BRCA-gene synthetic lethality and target PARP inhibitor resistance

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Diana Zatreanu ◽  
Helen M. R. Robinson ◽  
Omar Alkhatib ◽  
Marie Boursier ◽  
Harry Finch ◽  
...  
Keyword(s):  
Dna Repair ◽  
Synthetic Lethality ◽  
Parp Inhibitor ◽  
Repair Process ◽  
End Joining ◽  
Synthetic Lethal ◽  
Non Homologous End Joining ◽  
Inhibitor Resistance

AbstractTo identify approaches to target DNA repair vulnerabilities in cancer, we discovered nanomolar potent, selective, low molecular weight (MW), allosteric inhibitors of the polymerase function of DNA polymerase Polθ, including ART558. ART558 inhibits the major Polθ-mediated DNA repair process, Theta-Mediated End Joining, without targeting Non-Homologous End Joining. In addition, ART558 elicits DNA damage and synthetic lethality in BRCA1- or BRCA2-mutant tumour cells and enhances the effects of a PARP inhibitor. Genetic perturbation screening revealed that defects in the 53BP1/Shieldin complex, which cause PARP inhibitor resistance, result in in vitro and in vivo sensitivity to small molecule Polθ polymerase inhibitors. Mechanistically, ART558 increases biomarkers of single-stranded DNA and synthetic lethality in 53BP1-defective cells whilst the inhibition of DNA nucleases that promote end-resection reversed these effects, implicating these in the synthetic lethal mechanism-of-action. Taken together, these observations describe a drug class that elicits BRCA-gene synthetic lethality and PARP inhibitor synergy, as well as targeting a biomarker-defined mechanism of PARPi-resistance.

scholarly journals Multi-layered chromatin proteomics identifies cell vulnerabilities in DNA repair

2021 ◽  
Author(s):  
Gianluca Sigismondo ◽  
Lavinia Arseni ◽  
Thomas G Hofmann ◽  
Martina Seiffert ◽  
Jeroen Krijgsveld
Keyword(s):  
Genome Stability ◽  
Parp Inhibitor ◽  
Strand Break ◽  
Parp Inhibitors ◽  
High Temporal Resolution ◽  
End Joining ◽  
Post Translational Modifications ◽  
Dna Double Strand Break ◽  
Associated Proteins ◽  
Non Homologous End Joining

The DNA damage response (DDR) is essential to maintain genome stability, and its deregulation predisposes to carcinogenesis while encompassing attractive targets for cancer therapy. Chromatin governs the DDR via interplay among all chromatin layers including DNA, histones post-translational modifications (hPTMs), and chromatin-associated proteins. Here we employ multi-layered proteomics to characterize chromatin-mediated interactions of repair proteins, signatures of hPTMs, and the DNA-bound proteome during DNA double-strand break repair at high temporal resolution. We functionally attribute novel chromatin-associated proteins to repair by non-homologous end-joining or homologous recombination (HR) revealing histone reader ATAD2, microtubule organizer TPX2 and histone methyltransferase G9A as regulators of HR and PARP inhibitor sensitivity. Furthermore, we dynamically profile numerous hPTMs at γH2AX-mononucleosomes during the DDR. Integration of these complementary data implicated G9A-mediated monomethylation of H3K56 in HR. Collectively, we provide a dynamic chromatin-centered view of DDR, while representing a valuable resource for the use of PARP inhibitors in cancer.

scholarly journals Withanolide D Enhances Radiosensitivity of Human Cancer Cells by Inhibiting DNA Damage Non-homologous End Joining Repair Pathway

2020 ◽  
Vol 9 ◽  
Author(s):  
Jerome Lacombe ◽  
Titouan Cretignier ◽  
Laetitia Meli ◽  
E. M. Kithsiri Wijeratne ◽  
Jean-Luc Veuthey ◽  
...  
Keyword(s):  
Dna Damage ◽  
Cancer Cells ◽  
Human Cancer ◽  
Repair Pathway ◽  
End Joining ◽  
Human Cancer Cells ◽  
Non Homologous End Joining

scholarly journals Structural basis for proficient oxidized ribonucleotide insertion in double strand break repair

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Joonas A. Jamsen ◽  
Akira Sassa ◽  
Lalith Perera ◽  
David D. Shock ◽  
William A. Beard ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Time Lapse ◽  
Strand Break ◽  
Structural Basis ◽  
End Joining ◽  
Strand Breaks ◽  
Nucleotide Pools ◽  
Nucleotide Insertion ◽  
Non Homologous End Joining

AbstractReactive oxygen species (ROS) oxidize cellular nucleotide pools and cause double strand breaks (DSBs). Non-homologous end-joining (NHEJ) attaches broken chromosomal ends together in mammalian cells. Ribonucleotide insertion by DNA polymerase (pol) μ prepares breaks for end-joining and this is required for successful NHEJ in vivo. We previously showed that pol μ lacks discrimination against oxidized dGTP (8-oxo-dGTP), that can lead to mutagenesis, cancer, aging and human disease. Here we reveal the structural basis for proficient oxidized ribonucleotide (8-oxo-rGTP) incorporation during DSB repair by pol μ. Time-lapse crystallography snapshots of structural intermediates during nucleotide insertion along with computational simulations reveal substrate, metal and side chain dynamics, that allow oxidized ribonucleotides to escape polymerase discrimination checkpoints. Abundant nucleotide pools, combined with inefficient sanitization and repair, implicate pol μ mediated oxidized ribonucleotide insertion as an emerging source of widespread persistent mutagenesis and genomic instability.

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