scholarly journals Replicated chromatin curtails 53BP1 recruitment in BRCA1-proficient and BRCA1-deficient cells

2021 ◽  
Vol 4 (6) ◽  
pp. e202101023
Author(s):  
Jone Michelena ◽  
Stefania Pellegrino ◽  
Vincent Spegg ◽  
Matthias Altmeyer
Keyword(s):  
Homologous Recombination ◽  
Single Cell ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Homology Directed Repair ◽  
Non Homologous End Joining ◽  
Cellular Phenotypes ◽  
Template Dna ◽  
Cell Data

DNA double-strand breaks can be repaired by non-homologous end-joining or homologous recombination. Which pathway is used depends on the balance between the tumor suppressors 53BP1 and BRCA1 and on the availability of an undamaged template DNA for homology-directed repair. How cells switch from a 53BP1-dominated to a BRCA1-governed homologous recombination response as they progress through the cell cycle is incompletely understood. Here we reveal, using high-throughput microscopy and applying single cell normalization to control for increased genome size as cells replicate their DNA, that 53BP1 recruitment to damaged replicated chromatin is inefficient in both BRCA1-proficient and BRCA1-deficient cells. Our results substantiate a dual switch model from a 53BP1-dominated response in unreplicated chromatin to a BRCA1–BARD1–dominated response in replicated chromatin, in which replication-coupled dilution of 53BP1’s binding mark H4K20me2 functionally cooperates with BRCA1–BARD1–mediated suppression of 53BP1 binding. More generally, we suggest that appropriate normalization of single cell data, for example, to DNA content, provides additional layers of information, which can be critical for quantifying and interpreting cellular phenotypes.

scholarly journals Replicated chromatin curtails 53BP1 recruitment in BRCA1-proficient and -deficient cells

2020 ◽  
Author(s):  
Jone Michelena ◽  
Stefania Pellegrino ◽  
Vincent Spegg ◽  
Matthias Altmeyer
Keyword(s):  
Single Cell ◽  
Repair Pathway ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Homology Directed Repair ◽  
Dna End Resection ◽  
Non Homologous End Joining ◽  
Cellular Phenotypes ◽  
Template Dna

AbstractDNA double-strand breaks can be repaired by two competing mechanisms, non-homologous end-joining (NHEJ) and homologous recombination (HR). Whether one or the other repair pathway is favored depends on the availability of an undamaged template DNA that allows for homology-directed repair. The tumor suppressor proteins 53BP1 and BRCA1 are considered antagonistic players in this repair pathway choice, as 53BP1 restrains DNA end resection, whereas BRCA1, together with its partner protein BARD1, displaces 53BP1 from damaged replicated chromatin and promotes HR. How cells switch from a 53BP1-dominated to a BRCA1-dominated response as they progress through the cell cycle is incompletely understood. Here we reveal, using high-throughput microscopy and applying single cell normalization to control for increased genome size as cells replicate their DNA, that 53BP1 recruitment to damaged replicated chromatin is inefficient in both BRCA1-proficient and BRCA1-deficient cells, in comparison to 53BP1 accumulation at damaged unreplicated chromatin. These findings substantiate a dual switch model from a 53BP1-dominated response in unreplicated chromatin to a BRCA1-BARD1-dominated response in replicated chromatin, in which replication-coupled dilution of 53BP1’s binding mark H4K20me2 functionally cooperates with BRCA1-BARD1-mediated suppression of 53BP1 binding. More generally, we suggest that appropriate normalization of single cell data, e.g. to DNA content, provides additional layers of information, which can be critical for quantifying and interpreting cellular phenotypes.

scholarly journals Depletion of Akt1 and Akt2 Impairs the Repair of Radiation-Induced DNA Double Strand Breaks via Homologous Recombination

2019 ◽  
Vol 20 (24) ◽  
pp. 6316 ◽  
Author(s):  
Tahereh Mohammadian Gol ◽  
H. Peter Rodemann ◽  
Klaus Dittmann
Keyword(s):  
Homologous Recombination ◽  
Double Strand Breaks ◽  
Major Protein ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Knock Out ◽  
Strand Breaks ◽  
Protein Levels ◽  
Non Homologous End Joining

Homologous recombination repair (HRR), non-homologous end-joining (NHEJ) and alternative NHEJ are major pathways that are utilized by cells for processing DNA double strand breaks (DNA-DSBs); their function plays an important role in the radiation resistance of tumor cells. Conflicting data exist regarding the role of Akt in homologous recombination (HR), i.e., the regulation of Rad51 as a major protein of this pathway. This study was designed to investigate the specific involvement of Akt isoforms in HRR. HCT116 colon cancer cells with stable AKT-knock-out and siRNA-mediated AKT-knockdown phenotypes were used to investigate the role of Akt1 and Akt2 isoforms in HR. The results clearly demonstrated that HCT116 AKT1-KO and AKT2-KO cells have a significantly reduced Rad51 foci formation 6 h post irradiation versus parental cells. Depletion of Akt1 and Akt2 protein levels as well as inhibition of Akt kinase activity resulted in an increased number of residual-γH2AX in CENP-F positive cells mainly representing the S and G2 phase cells. Furthermore, inhibition of NHEJ and HR using DNA-PK and Rad51 antagonists resulted in stronger radiosensitivity of AKT1 and AKT2 knockout cells versus wild type cells. These data collectively show that both Akt1 and Akt2 are involved in DSBs repair through HRR.

scholarly journals Homology Directed Repair by Cas9:Donor Co-localization in Mammalian Cells

2018 ◽  
Author(s):  
Philip J.R. Roche ◽  
Heidi Gytz ◽  
Faiz Hussain ◽  
Christopher J.F. Cameron ◽  
Denis Paquette ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Gene Editing ◽  
Low Frequency ◽  
Cost Effective ◽  
Hek293 Cells ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Homology Directed Repair ◽  
Non Homologous End Joining

AbstractHomology directed repair (HDR) induced by site specific DNA double strand breaks (DSB) with CRISPR/Cas9 is a precision gene editing approach that occurs at low frequency in comparison to indel forming non homologous end joining (NHEJ). In order to obtain high HDR percentages in mammalian cells, we engineered Cas9 protein fused to a high-affinity monoavidin domain to deliver biotinylated donor DNA to a DSB site. In addition, we used the cationic polymer, polyethylenimine, to deliver Cas9 RNP-donor DNA complex into the cell. Combining these strategies improved HDR percentages of up to 90% in three tested loci (CXCR4, EMX1, and TLR) in standard HEK293 cells. Our approach offers a cost effective, simple and broadly applicable gene editing method, thereby expanding the CRISPR/Cas9 genome editing toolbox.SummaryPrecision gene editing occurs at a low percentage in mammalian cells using Cas9. Colocalization of donor with Cas9MAV and PEI delivery raises HDR occurrence.

scholarly journals The DNAPKcs long-range C-NHEJ complex is required for blunt DNA end joining when XLF is weakened

2021 ◽  
Author(s):  
Metztli Cisneros-Aguirre ◽  
Felicia Wednesday Lopezcolorado ◽  
Linda Jillianne Tsai ◽  
Ragini Bhargava ◽  
Jeremy M Stark
Keyword(s):  
Long Range ◽  
De Novo ◽  
Dna Double Strand Breaks ◽  
Genome Maintenance ◽  
End Joining ◽  
Strand Breaks ◽  
Homology Directed Repair ◽  
Non Homologous End Joining ◽  
Dna Ends

Canonical non-homologous end joining (C-NHEJ) factors can assemble into a long-range (LR) complex with DNA ends relatively far apart that contains DNAPKcs, XLF, XRCC4, LIG4, and the KU heterodimer and a short-range (SR) complex lacking DNAPKcs that has the ends positioned for ligation. Since the SR complex can form de novo, the role of the LR complex (i.e., DNAPKcs) for chromosomal EJ is unclear. We have examined EJ of chromosomal blunt DNA double-strand breaks (DSBs), and found that DNAPKcs is significantly less important than XLF and XRCC4 for such EJ. However, weakening XLF via disrupting interaction interfaces (e.g., disrupting the XLF homodimer interface) causes a marked requirement for DNAPKcs, its kinase activity, and its ABCDE-cluster autophosphorylation sites for blunt DSB EJ. In contrast, other aspects of genome maintenance are sensitive to DNAPKcs kinase inhibition in a manner that is not further enhanced by XLF loss (i.e., suppression of homology-directed repair and structural variants, and IR-resistance). We suggest that DNAPKcs is required to position a weakened XLF in an LR complex that can transition into a functional SR complex for blunt DSB EJ, but also has distinct functions for other aspects of genome maintenance.

scholarly journals MiCas9 increases large size gene knock-in rates and reduces undesirable on-target and off-target indel edits

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Linyuan Ma ◽  
Jinxue Ruan ◽  
Jun Song ◽  
Luan Wen ◽  
Dongshan Yang ◽  
...  
Keyword(s):  
Gene Editing ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Repair Capacity ◽  
Strand Breaks ◽  
Homology Directed Repair ◽  
Insertion And Deletion ◽  
Large Size ◽  
Target Locus ◽  
Non Homologous End Joining

AbstractGene editing nuclease represented by Cas9 efficiently generates DNA double strand breaks at the target locus, followed by repair through either the error-prone non-homologous end joining or the homology directed repair pathways. To improve Cas9’s homology directed repair capacity, here we report the development of miCas9 by fusing a minimal motif consisting of thirty-six amino acids to spCas9. MiCas9 binds RAD51 through this fusion motif and enriches RAD51 at the target locus. In comparison to spCas9, miCas9 enhances double-stranded DNA mediated large size gene knock-in rates, systematically reduces off-target insertion and deletion events, maintains or increases single-stranded oligodeoxynucleotides mediated precise gene editing rates, and effectively reduces on-target insertion and deletion rates in knock-in applications. Furthermore, we demonstrate that this fusion motif can work as a “plug and play” module, compatible and synergistic with other Cas9 variants. MiCas9 and the minimal fusion motif may find broad applications in gene editing research and therapeutics.

scholarly journals Covalent linkage of the DNA repair template to the CRISPR/Cas9 complex enhances homology-directed repair

2017 ◽  
Author(s):  
Natasa Savic ◽  
Femke Ringnalda ◽  
Katja Bargsten ◽  
Yizhou Li ◽  
Christian Berk ◽  
...  
Keyword(s):  
Dna Repair ◽  
Mammalian Cells ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Homology Directed Repair ◽  
Genetic Modifications ◽  
Repair Template ◽  
Single Base Pair ◽  
Non Homologous End Joining

AbstractThe CRISPR/Cas9 targeted nuclease technology allows the insertion of genetic modifications with single base-pair precision. The preference of mammalian cells to repair Cas9-induced DNA double-strand breaks via non-homologous end joining (NHEJ) rather than via homology-directed repair (HDR) however leads to relatively low rates of correctly edited loci. Here we demonstrate that covalently linking the DNA repair template to Cas9 increases the ratio of HDR over NHEJ up to 23-fold, and therefore provides advantages for clinical applications where high-fidelity repair is needed.

scholarly journals DNA-dependent protein kinase promotes DNA end processing by MRN and CtIP

2018 ◽  
Author(s):  
Rajashree A. Deshpande ◽  
Logan R. Myler ◽  
Michael M. Soniat ◽  
Nodar Makharashvili ◽  
Linda Lee ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Homologous Recombination ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Dependent Protein Kinase ◽  
Strand Breaks ◽  
Mrn Complex ◽  
Dependent Protein ◽  
Non Homologous End Joining ◽  
Dna Ends

AbstractThe repair of DNA double-strand breaks occurs through non-homologous end joining or homologous recombination in vertebrate cells - a choice that is thought to be decided by a competition between DNA-dependent protein kinase (DNA-PK) and the Mre11/Rad50/Nbs1 (MRN) complex but is not well understood. Using ensemble biochemistry and single-molecule approaches, here we show that the MRN complex is dependent on DNA-PK and phosphorylated CtIP to perform efficient processing and resection of DNA ends in physiological conditions, thus eliminating the competition model. Endonucleolytic removal of DNA-PK-bound DNA ends is also observed at double-strand break sites in human cells. The involvement of DNA-PK in MRN-mediated end processing promotes an efficient and sequential transition from non-homologous end joining to homologous recombination by facilitating DNA-PK removal.One Sentence SummaryDNA-dependent protein kinase, an enzyme critical for non-homologous repair of DNA double-strand breaks, also stimulates end processing for homologous recombination.

Sign in / Sign up

Export Citation Format

Share Document