scholarly journals Mammalian polymerase θ promotes alternative NHEJ and suppresses recombination

Nature ◽  
2015 ◽  
Vol 518 (7538) ◽  
pp. 254-257 ◽  
Author(s):  
Pedro A. Mateos-Gomez ◽  
Fade Gong ◽  
Nidhi Nair ◽  
Kyle M. Miller ◽  
Eros Lazzerini-Denchi ◽  
...  
Keyword(s):  
Alternative Nhej

scholarly journals Alternative NHEJ Pathway Components Are Therapeutic Targets in High-Risk Neuroblastoma

2015 ◽  
Vol 13 (3) ◽  
pp. 470-482 ◽  
Author(s):  
Erika A. Newman ◽  
Fujia Lu ◽  
Daniela Bashllari ◽  
Li Wang ◽  
Anthony W. Opipari ◽  
...  
Keyword(s):  
High Risk ◽  
Therapeutic Targets ◽  
Nhej Pathway ◽  
Alternative Nhej

scholarly journals Biasing genome-editing events toward precise length deletions with an RNA-guided TevCas9 dual nuclease

2016 ◽  
Vol 113 (52) ◽  
pp. 14988-14993 ◽  
Author(s):  
Jason M. Wolfs ◽  
Thomas A. Hamilton ◽  
Jeremy T. Lant ◽  
Marcon Laforet ◽  
Jenny Zhang ◽  
...  
Keyword(s):  
Genome Editing ◽  
Homing Endonuclease ◽  
Target Site ◽  
Hek293 Cells ◽  
Dna Breaks ◽  
End Joining ◽  
Minimal Processing ◽  
Gene Knockouts ◽  
Alternative Nhej ◽  
Dna Ends

The CRISPR/Cas9 nuclease is commonly used to make gene knockouts. The blunt DNA ends generated by cleavage can be efficiently ligated by the classical nonhomologous end-joining repair pathway (c-NHEJ), regenerating the target site. This repair creates a cycle of cleavage, ligation, and target site regeneration that persists until sufficient modification of the DNA break by alternative NHEJ prevents further Cas9 cutting, generating a heterogeneous population of insertions and deletions typical of gene knockouts. Here, we develop a strategy to escape this cycle and bias events toward defined length deletions by creating an RNA-guided dual active site nuclease that generates two noncompatible DNA breaks at a target site, effectively deleting the majority of the target site such that it cannot be regenerated. The TevCas9 nuclease, a fusion of the I-TevI nuclease domain to Cas9, functions robustly in HEK293 cells and generates 33- to 36-bp deletions at frequencies up to 40%. Deep sequencing revealed minimal processing of TevCas9 products, consistent with protection of the DNA ends from exonucleolytic degradation and repair by the c-NHEJ pathway. Directed evolution experiments identified I-TevI variants with broadened targeting range, making TevCas9 an easy-to-use reagent. Our results highlight how the sequence-tolerant cleavage properties of the I-TevI homing endonuclease can be harnessed to enhance Cas9 applications, circumventing the cleavage and ligation cycle and biasing genome-editing events toward defined length deletions.

scholarly journals Repair of G1 induced DNA double-strand breaks in S-G2/M by alternative NHEJ

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Wei Yu ◽  
Chloé Lescale ◽  
Loelia Babin ◽  
Marie Bedora-Faure ◽  
Hélène Lenden-Hasse ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Strand Break ◽  
Cancer Therapies ◽  
Dna Double Strand Breaks ◽  
Dna Breaks ◽  
Dsb Repair ◽  
Chromosome Translocations ◽  
Dna End Resection ◽  
G1 Cells ◽  
Alternative Nhej

Abstract The alternative non-homologous end-joining (NHEJ) pathway promotes DNA double-strand break (DSB) repair in cells deficient for NHEJ or homologous recombination, suggesting that it operates at all stages of the cell cycle. Here, we use an approach in which DNA breaks can be induced in G1 cells and their repair tracked, enabling us to show that joining of DSBs is not functional in G1-arrested XRCC4-deficient cells. Cell cycle entry into S-G2/M restores DSB repair by Pol θ-dependent and PARP1-independent alternative NHEJ with repair products bearing kilo-base long DNA end resection, micro-homologies and chromosome translocations. We identify a synthetic lethal interaction between XRCC4 and Pol θ under conditions of G1 DSBs, associated with accumulation of unresolved DNA ends in S-G2/M. Collectively, our results support the conclusion that the repair of G1 DSBs progressing to S-G2/M by alternative NHEJ drives genomic instability and represent an attractive target for future DNA repair-based cancer therapies.

scholarly journals Alternative-NHEJ Is a Mechanistically Distinct Pathway of Mammalian Chromosome Break Repair

PLoS Genetics ◽  
2008 ◽  
Vol 4 (6) ◽  
pp. e1000110 ◽  
Author(s):  
Nicole Bennardo ◽  
Anita Cheng ◽  
Nick Huang ◽  
Jeremy M. Stark
Keyword(s):  
Chromosome Break ◽  
Break Repair ◽  
Alternative Nhej

Effects of chromatin decondensation on alternative NHEJ

DNA Repair ◽  
2013 ◽  
Vol 12 (11) ◽  
pp. 972-981 ◽  
Author(s):  
Mario Moscariello ◽  
George Iliakis
Keyword(s):  
Chromatin Decondensation ◽  
Alternative Nhej

scholarly journals Parp1 facilitates alternative NHEJ, whereas Parp2 suppresses IgH/c-myc translocations during immunoglobulin class switch recombination

2009 ◽  
Vol 206 (5) ◽  
pp. 1047-1056 ◽  
Author(s):  
Isabelle Robert ◽  
Françoise Dantzer ◽  
Bernardo Reina-San-Martin
Keyword(s):  
Dna Damage ◽  
Cytidine Deaminase ◽  
Class Switch Recombination ◽  
Dependent Manner ◽  
Dna Breaks ◽  
End Joining ◽  
Immunoglobulin Class ◽  
Class Switch ◽  
Switch Regions ◽  
Alternative Nhej

Immunoglobulin class switch recombination (CSR) is initiated by DNA breaks triggered by activation-induced cytidine deaminase (AID). These breaks activate DNA damage response proteins to promote appropriate repair and long-range recombination. Aberrant processing of these breaks, however, results in decreased CSR and/or increased frequency of illegitimate recombination between the immunoglobulin heavy chain locus and oncogenes like c-myc. Here, we have examined the contribution of the DNA damage sensors Parp1 and Parp2 in the resolution of AID-induced DNA breaks during CSR. We find that although Parp enzymatic activity is induced in an AID-dependent manner during CSR, neither Parp1 nor Parp2 are required for CSR. We find however, that Parp1 favors repair of switch regions through a microhomology-mediated pathway and that Parp2 actively suppresses IgH/c-myc translocations. Thus, we define Parp1 as facilitating alternative end-joining and Parp2 as a novel translocation suppressor during CSR.

scholarly journals Polo-like kinase 3 regulates CtIP during DNA double-strand break repair in G1

2014 ◽  
Vol 206 (7) ◽  
pp. 877-894 ◽  
Author(s):  
Olivia Barton ◽  
Steffen C. Naumann ◽  
Ronja Diemer-Biehs ◽  
Julia Künzel ◽  
Monika Steinlage ◽  
...  
Keyword(s):  
Large Scale ◽  
Strand Break ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Interacting Protein ◽  
Genomic Deletions ◽  
Cellular Processes ◽  
Dna Double Strand Break ◽  
Radiation Induced ◽  
Alternative Nhej

DNA double-strand breaks (DSBs) are repaired by nonhomologous end joining (NHEJ) or homologous recombination (HR). The C terminal binding protein–interacting protein (CtIP) is phosphorylated in G2 by cyclin-dependent kinases to initiate resection and promote HR. CtIP also exerts functions during NHEJ, although the mechanism phosphorylating CtIP in G1 is unknown. In this paper, we identify Plk3 (Polo-like kinase 3) as a novel DSB response factor that phosphorylates CtIP in G1 in a damage-inducible manner and impacts on various cellular processes in G1. First, Plk3 and CtIP enhance the formation of ionizing radiation-induced translocations; second, they promote large-scale genomic deletions from restriction enzyme-induced DSBs; third, they are required for resection and repair of complex DSBs; and finally, they regulate alternative NHEJ processes in Ku−/− mutants. We show that mutating CtIP at S327 or T847 to nonphosphorylatable alanine phenocopies Plk3 or CtIP loss. Plk3 binds to CtIP phosphorylated at S327 via its Polo box domains, which is necessary for robust damage-induced CtIP phosphorylation at S327 and subsequent CtIP phosphorylation at T847.

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