scholarly journals A high-throughput, flow cytometry-based method to quantify DNA-end resection in mammalian cells

2012 ◽  
Vol 81A (10) ◽  
pp. 922-928 ◽  
Author(s):  
Josep V. Forment ◽  
Rachael V. Walker ◽  
Stephen P. Jackson
Keyword(s):  
Flow Cytometry ◽  
High Throughput ◽  
Mammalian Cells ◽  
Dna End Resection ◽  
End Resection

scholarly journals DNA-PK Promotes DNA End Resection at DNA Double Strand Breaks in G0 cells

2021 ◽  
Author(s):  
Jessica K Tyler ◽  
Faith C Fowler ◽  
Chelsea Peart ◽  
Bo-Ruei Chen ◽  
Barry Sleckman ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Mammalian Cells ◽  
Strand Break ◽  
Dna Double Strand Breaks ◽  
Proliferating Cells ◽  
Dsb Repair ◽  
Extensive Resection ◽  
Key Factor ◽  
Dna End Resection ◽  
End Resection

DNA double-strand break (DSB) repair by homologous recombination is confined to the S and G2 phases of the cell cycle partly due to 53BP1 antagonizing DNA end resection in G1 phase and non-cycling quiescent (G0) cells where DSBs are predominately repaired by non-homologous end joining (NHEJ). Unexpectedly, we uncovered extensive MRE11- and CtIP-dependent DNA end resection at DSBs in G0 mammalian cells. A whole genome CRISPR/Cas9 screen revealed the DNA-dependent kinase (DNA-PK) complex as a key factor in promoting DNA end resection in G0 cells. In agreement, depletion of FBXL12, which promotes ubiquitylation and removal of the KU70/KU80 subunits of DNA-PK from DSBs, promotes even more extensive resection in G0 cells. In contrast, a requirement for DNA-PK in promoting DNA end resection in proliferating cells at the G1 or G2 phase of the cell cycle was not observed. Our findings establish that DNA-PK uniquely promotes DNA end resection in G0, but not in G1 or G2 phase cells, and has important implications for DNA DSB repair in quiescent cells.

scholarly journals DNA end resection and its role in DNA replication and DSB repair choice in mammalian cells

2020 ◽  
Vol 52 (10) ◽  
pp. 1705-1714 ◽  
Author(s):  
Fei Zhao ◽  
Wootae Kim ◽  
Jake A. Kloeber ◽  
Zhenkun Lou
Keyword(s):  
Dna Repair ◽  
Dna Replication ◽  
Mammalian Cells ◽  
Strand Break ◽  
Double Strand Break ◽  
Double Strand Break Repair ◽  
General Process ◽  
Break Repair ◽  
Dna End Resection ◽  
End Resection

Abstract DNA end resection has a key role in double-strand break repair and DNA replication. Defective DNA end resection can cause malfunctions in DNA repair and replication, leading to greater genomic instability. DNA end resection is initiated by MRN-CtIP generating short, 3′-single-stranded DNA (ssDNA). This newly generated ssDNA is further elongated by multiple nucleases and DNA helicases, such as EXO1, DNA2, and BLM. Effective DNA end resection is essential for error-free homologous recombination DNA repair, the degradation of incorrectly replicated DNA and double-strand break repair choice. Because of its importance in DNA repair, DNA end resection is strictly regulated. Numerous mechanisms have been reported to regulate the initiation, extension, and termination of DNA end resection. Here, we review the general process of DNA end resection and its role in DNA replication and repair pathway choice.

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.

A deep learning-based concept for high throughput image flow cytometry

2021 ◽  
Vol 118 (12) ◽  
pp. 123701
Author(s):  
Julie Martin-Wortham ◽  
Steffen M. Recktenwald ◽  
Marcelle G. M. Lopes ◽  
Lars Kaestner ◽  
Christian Wagner ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Deep Learning ◽  
High Throughput ◽  
Image Flow

scholarly journals High-throughput multiparametric imaging flow cytometry: toward diffraction-limited sub-cellular detection and monitoring of sub-cellular processes

Cell Reports ◽  
2021 ◽  
Vol 34 (10) ◽  
pp. 108824
Author(s):  
Gregor Holzner ◽  
Bogdan Mateescu ◽  
Daniel van Leeuwen ◽  
Gea Cereghetti ◽  
Reinhard Dechant ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
High Throughput ◽  
Multiparametric Imaging ◽  
Cellular Processes ◽  
Imaging Flow Cytometry

scholarly journals DHX9-dependent recruitment of BRCA1 to RNA promotes DNA end resection in homologous recombination

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Prasun Chakraborty ◽  
Kevin Hiom
Keyword(s):  
Dna Damage ◽  
Homologous Recombination ◽  
Rna Polymerase Ii ◽  
Critical Role ◽  
Dna Breaks ◽  
Double Stranded Dna ◽  
Recombination Repair ◽  
Dna End Resection ◽  
End Resection ◽  
Rna Polymerase Ii Transcription

AbstractDouble stranded DNA Breaks (DSB) that occur in highly transcribed regions of the genome are preferentially repaired by homologous recombination repair (HR). However, the mechanisms that link transcription with HR are unknown. Here we identify a critical role for DHX9, a RNA helicase involved in the processing of pre-mRNA during transcription, in the initiation of HR. Cells that are deficient in DHX9 are impaired in the recruitment of RPA and RAD51 to sites of DNA damage and fail to repair DSB by HR. Consequently, these cells are hypersensitive to treatment with agents such as camptothecin and Olaparib that block transcription and generate DSB that specifically require HR for their repair. We show that DHX9 plays a critical role in HR by promoting the recruitment of BRCA1 to RNA as part of the RNA Polymerase II transcription complex, where it facilitates the resection of DSB. Moreover, defects in DHX9 also lead to impaired ATR-mediated damage signalling and an inability to restart DNA replication at camptothecin-induced DSB. Together, our data reveal a previously unknown role for DHX9 in the DNA Damage Response that provides a critical link between RNA, RNA Pol II and the repair of DNA damage by homologous recombination.

scholarly journals A “No‐Touch” Antibody‐Staining Method of Adherent Cells for High‐Throughput Flow Cytometry in 384‐Well Microplate Format for Cell‐Based Drug Library Screening

2019 ◽  
Vol 97 (8) ◽  
pp. 845-851 ◽  
Author(s):  
Annelisa M. Cornel ◽  
Celina L. Szanto ◽  
Niek P. Til ◽  
Jeroen F. Velzen ◽  
Jaap J. Boelens ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
High Throughput ◽  
Staining Method ◽  
Adherent Cells ◽  
Library Screening ◽  
Antibody Staining
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