The repair of topoisomerase 2 cleavage complexes in Arabidopsis

2021 ◽  
Author(s):  
Leonie Hacker ◽  
Annika Dorn ◽  
Janina Enderle ◽  
Holger Puchta
Keyword(s):  
Genome Stability ◽  
Nonhomologous End Joining ◽  
Plant Genome ◽  
Minor Role ◽  
End Joining ◽  
Induced Mutants ◽  
Topoisomerase 2 ◽  
A Minor ◽  
Alternative Nhej ◽  
Dna Ends

Abstract DNA–protein crosslinks (DPCs) and DNA double-stranded breaks (DSBs), including those produced by stalled topoisomerase 2 cleavage complexes (TOP2ccs), must be repaired to ensure genome stability. The basic mechanisms of TOP2cc repair have been characterized in other eukaryotes, but we lack information for plants. Using CRISPR/Cas-induced mutants, we show that Arabidopsis thaliana has two main TOP2cc repair pathways: one is defined by TYROSYL-DNA-PHOSPHODIESTERASE 2 (TDP2), which hydrolyzes TOP2–DNA linkages, the other by the DNA-dependent protease WSS1A (a homolog of human SPARTAN/yeast weak suppressor of smt3 [Wss1]), which also functions in DPC repair. TDP1 and TDP2 function nonredundantly in TOP1cc repair, indicating that they act specifically on their respective stalled cleavage complexes. The nuclease METHYL METHANESULFONATE AND UV-SENSITIVE PROTEIN 81 (MUS81) plays a major role in global DPC repair and a minor role in TOP2cc repair. DSBs arise as intermediates of TOP2cc repair and are repaired by classical and alternative nonhomologous end joining (NHEJ) pathways. Double-mutant analysis indicates that “clean” DNA ends caused by TDP2 hydrolysis are mainly religated by classical NHEJ, which helps avoid mutation. In contrast, the mutagenic alternative NHEJ pathway mainly processes nonligateable DNA ends. Thus, TDP2 promotes maintenance of plant genome integrity by error-free repair of TOP2cc.

Repair of DNA Double-Strand Breaks by the Nonhomologous End Joining Pathway

2021 ◽  
Vol 90 (1) ◽  
Author(s):  
Benjamin M. Stinson ◽  
Joseph J. Loparo
Keyword(s):  
Genome Stability ◽  
Double Strand Breaks ◽  
Nonhomologous End Joining ◽  
Annual Review ◽  
Publication Date ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Break Site ◽  
Dna Ends

DNA double-strand breaks pose a serious threat to genome stability. In vertebrates, these breaks are predominantly repaired by nonhomologous end joining (NHEJ), which pairs DNA ends in a multiprotein synaptic complex to promote their direct ligation. NHEJ is a highly versatile pathway that uses an array of processing enzymes to modify damaged DNA ends and enable their ligation. The mechanisms of end synapsis and end processing have important implications for genome stability. Rapid and stable synapsis is necessary to limit chromosome translocations that result from the mispairing of DNA ends. Furthermore, end processing must be tightly regulated to minimize mutations at the break site. Here, we review our current mechanistic understanding of vertebrate NHEJ, with a particular focus on end synapsis and processing. Expected final online publication date for the Annual Review of Biochemistry, Volume 90 is June 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

scholarly journals Rearrangements of the DNA in Carbon Ion-Induced Mutants of Arabidopsis thaliana

Genetics ◽  
2001 ◽  
Vol 157 (1) ◽  
pp. 379-387 ◽  
Author(s):  
Naoya Shikazono ◽  
Atsushi Tanaka ◽  
Hiroshi Watanabe ◽  
Shigemitsu Tano
Keyword(s):  
Arabidopsis Thaliana ◽  
Nonhomologous End Joining ◽  
Chromosome 3 ◽  
Chromosome 2 ◽  
Carbon Ions ◽  
Induced Mutations ◽  
End Joining ◽  
Carbon Ion ◽  
Induced Mutants ◽  
Dna Strand

Abstract To elucidate the nature of structural alterations in plants, three carbon ion-induced mutations in Arabidopsis thaliana, gl1-3, tt4(C1), and ttg1-21, were analyzed. The gl1-3 mutation was found to be generated by an inversion of a fragment that contained GL1 and Atpk7 loci on chromosome 3. The size of the inverted fragment was a few hundred kilobase pairs. The inversion was found to accompany an insertion of a 107-bp fragment derived from chromosome 2. The tt4(C1) mutation was also found to be due to an inversion. The size of the intervening region between the breakpoints was also estimated to be a few hundred kilobase pairs. In the case of ttg1-21, it was found that a break occurred at the TTG1 locus on chromosome 5, and reciprocal translocation took place between it and chromosome 3. From the sequences flanking the breakpoints, the DNA strand breaks induced by carbon ions were found to be rejoined using, if present, only short homologous sequences. Small deletions were also observed around the breakpoints. These results suggest that the nonhomologous end-joining (NHEJ) pathway operates after plant cells are exposed to ion particles.

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 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 Loading of the Nonhomologous End Joining Factor, Ku, on Protein-occluded DNA Ends

2007 ◽  
Vol 282 (14) ◽  
pp. 10605-10613 ◽  
Author(s):  
Steven A. Roberts ◽  
Dale A. Ramsden
Keyword(s):  
Nonhomologous End Joining ◽  
End Joining ◽  
Dna Ends

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.

DNA End Resection: Mechanism and Control

2021 ◽  
Vol 55 (1) ◽  
pp. 285-307
Author(s):  
Petr Cejka ◽  
Lorraine S. Symington
Keyword(s):  
Double Strand Breaks ◽  
Nonhomologous End Joining ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Critical Determinant ◽  
Strand Breaks ◽  
Dna End Resection ◽  
And Control ◽  
End Resection ◽  
Dna Ends

DNA double-strand breaks (DSBs) are cytotoxic lesions that threaten genome integrity and cell viability. Typically, cells repair DSBs by either nonhomologous end joining (NHEJ) or homologous recombination (HR). The relative use of these two pathways depends on many factors, including cell cycle stage and the nature of the DNA ends. A critical determinant of repair pathway selection is the initiation of 5′→3′ nucleolytic degradation of DNA ends, a process referred to as DNA end resection. End resection is essential to create single-stranded DNA overhangs, which serve as the substrate for the Rad51 recombinase to initiate HR and are refractory to NHEJ repair. Here, we review recent insights into the mechanisms of end resection, how it is regulated, and the pathological consequences of its dysregulation.

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