scholarly journals An Efficient Method to Generate Chromosomal Rearrangements by Targeted DNA Double-Strand Breaks in Drosophila melanogaster

2004 ◽  
Vol 14 (7) ◽  
pp. 1382-1393 ◽  
Author(s):  
D. Egli
Keyword(s):  
Drosophila Melanogaster ◽  
Efficient Method ◽  
Chromosomal Rearrangements ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks

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 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.

DNA double-strand breaks, chromosomal rearrangements, and genomic instability

1998 ◽  
Vol 404 (1-2) ◽  
pp. 125-128 ◽  
Author(s):  
William F. Morgan ◽  
James Corcoran ◽  
Andreas Hartmann ◽  
Mark I. Kaplan ◽  
Charles L. Limoli ◽  
...  
Keyword(s):  
Genomic Instability ◽  
Chromosomal Rearrangements ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks

scholarly journals DNA Double-Strand Breaks Affect Chromosomal Rearrangements during Methotrexate-Mediated Gene Amplification in Chinese Hamster Ovary Cells

Pharmaceutics ◽  
2021 ◽  
Vol 13 (3) ◽  
pp. 376
Author(s):  
Jong Youn Baik ◽  
Hye-Jin Han ◽  
Kelvin H. Lee
Keyword(s):  
Cell Lines ◽  
Gene Amplification ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chromosomal Rearrangements ◽  
Chinese Hamster ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
The Impact

Methotrexate (MTX)-mediated gene amplification has been widely used in Chinese hamster ovary (CHO) cells for the biomanufacturing of therapeutic proteins. Although many studies have reported chromosomal instability and extensive chromosomal rearrangements in MTX-mediated gene-amplified cells, which may be associated with cell line instability issues, the mechanisms of chromosomal rearrangement formation remain poorly understood. We tested the impact of DNA double-strand breaks (DSBs) on chromosomal rearrangements using bleomycin, a DSB-inducing reagent. Bleomycin-treated CHO-DUK cells, which are one of the host cell lines deficient in dihydrofolate reductase (Dhfr) activity, exhibited a substantial number of cells containing radial formations or non-radial formations with chromosomal rearrangements, suggesting that DSBs may be associated with chromosomal rearrangements. To confirm the causes of DSBs during gene amplification, we tested the effects of MTX treatment and the removal of nucleotide base precursors on DSB formation in Dhfr-deficient (i.e., CHO-DUK) and Dhfr-expressing (i.e., CHO-K1) cells. Immunocytochemistry demonstrated that MTX treatment did not induce DSBs per se, but a nucleotide shortage caused by the MTX-mediated inhibition of Dhfr activity resulted in DSBs. Our data suggest that a nucleotide shortage caused by MTX-mediated Dhfr inhibition in production cell lines is the primary cause of a marked increase in DSBs, resulting in extensive chromosomal rearrangements after gene amplification processes.

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.

Repair pathway choice for double-strand breaks

2020 ◽  
Vol 64 (5) ◽  
pp. 765-777 ◽  
Author(s):  
Yixi Xu ◽  
Dongyi Xu
Keyword(s):  
Cell Cycle ◽  
Double Strand Breaks ◽  
Homologous Region ◽  
Gene Repair ◽  
Repair Pathway ◽  
Dna Double Strand Breaks ◽  
Environmental Radiation ◽  
Strand Breaks ◽  
Dna End Resection ◽  
End Resection

Abstract Deoxyribonucleic acid (DNA) is at a constant risk of damage from endogenous substances, environmental radiation, and chemical stressors. DNA double-strand breaks (DSBs) pose a significant threat to genomic integrity and cell survival. There are two major pathways for DSB repair: nonhomologous end-joining (NHEJ) and homologous recombination (HR). The extent of DNA end resection, which determines the length of the 3′ single-stranded DNA (ssDNA) overhang, is the primary factor that determines whether repair is carried out via NHEJ or HR. NHEJ, which does not require a 3′ ssDNA tail, occurs throughout the cell cycle. 53BP1 and the cofactors PTIP or RIF1-shieldin protect the broken DNA end, inhibit long-range end resection and thus promote NHEJ. In contrast, HR mainly occurs during the S/G2 phase and requires DNA end processing to create a 3′ tail that can invade a homologous region, ensuring faithful gene repair. BRCA1 and the cofactors CtIP, EXO1, BLM/DNA2, and the MRE11–RAD50–NBS1 (MRN) complex promote DNA end resection and thus HR. DNA resection is influenced by the cell cycle, the chromatin environment, and the complexity of the DNA end break. Herein, we summarize the key factors involved in repair pathway selection for DSBs and discuss recent related publications.

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