scholarly journals Accurate Homologous Recombination Is a Prominent Double-Strand Break Repair Pathway in Mammalian Chromosomes and Is Modulated by Mismatch Repair Protein Msh2

2007 ◽  
Vol 27 (22) ◽  
pp. 7816-7827 ◽  
Author(s):  
Jason A. Smith ◽  
Laura A. Bannister ◽  
Vikram Bhattacharjee ◽  
Yibin Wang ◽  
Barbara Criscuolo Waldman ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Cell Lines ◽  
Fusion Gene ◽  
Sequence Divergence ◽  
Strand Break ◽  
Double Strand Break ◽  
Repair Pathway ◽  
Dsb Repair ◽  
Spontaneous Recombination ◽  
Mammalian Cell Lines

ABSTRACT We designed DNA substrates to study intrachromosomal recombination in mammalian chromosomes. Each substrate contains a thymidine kinase (tk) gene fused to a neomycin resistance (neo) gene. The fusion gene is disrupted by an oligonucleotide containing the 18-bp recognition site for endonuclease I-SceI. Substrates also contain a “donor” tk sequence that displays 1% or 19% sequence divergence relative to the tk portion of the fusion gene. Each donor serves as a potential recombination partner for the fusion gene. After stably transfecting substrates into mammalian cell lines, we investigated spontaneous recombination and double-strand break (DSB)-induced recombination following I-SceI expression. No recombination events between sequences with 19% divergence were recovered. Strikingly, even though no selection for accurate repair was imposed, accurate conservative homologous recombination was the predominant DSB repair event recovered from rodent and human cell lines transfected with the substrate containing sequences displaying 1% divergence. Our work is the first unequivocal demonstration that homologous recombination can serve as a major DSB repair pathway in mammalian chromosomes. We also found that Msh2 can modulate homologous recombination in that Msh2 deficiency promoted discontinuity and increased length of gene conversion tracts and brought about a severalfold increase in the overall frequency of DSB-induced recombination.

scholarly journals A unique pathway of double-strand break repair operates in tandemly repeated genes.

1991 ◽  
Vol 11 (3) ◽  
pp. 1222-1231 ◽  
Author(s):  
B A Ozenberger ◽  
G S Roeder
Keyword(s):  
Gene Product ◽  
Gene Array ◽  
Strand Break ◽  
Double Strand Break ◽  
Dsb Repair ◽  
Yeast Saccharomyces Cerevisiae ◽  
Spontaneous Recombination ◽  
Genome Recombination ◽  
Repeat Units ◽  
Almost All

The RAD52 gene product of the yeast Saccharomyces cerevisiae is required for most spontaneous recombination and almost all double-strand break (DSB) repair. In contrast to recombination elsewhere in the genome, recombination in the ribosomal DNA (rDNA) array is RAD52 independent. To determine the fate of a DSB in the rDNA gene array, a cut site for the HO endonuclease was inserted into the rDNA in a strain containing an inducible HO gene. DSBs were efficiently repaired at this site, even in the absence of the RAD52 gene product. Efficient RAD52-independent DSB repair was also observed at another tandem gene array, CUP1, consisting of 18 repeat units. However, in a smaller CUP1 array, consisting of only three units, most DSBs (ca. 80%) were not repaired and resulted in cell death. All RAD52-independent DSB repair events examined resulted in the loss of one or more repeat units. We propose a model for DSB repair in repeated sequences involving the generation of single-stranded tails followed by reannealing.

scholarly journals The deSUMOylase SENP2 coordinates homologous recombination and non-homologous end joining by independent mechanisms

2018 ◽  
Author(s):  
Alexander J. Garvin ◽  
Alexandra K. Walker ◽  
Ruth M. Densham ◽  
Anoop Singh Chauhan ◽  
Helen R. Stone ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Strand Break ◽  
Double Strand Break ◽  
End Joining ◽  
Dsb Repair ◽  
Dna Double Strand Break ◽  
Non Homologous End Joining

AbstractSUMOylation in the DNA double-strand break (DSB) response regulates recruitment, activity and clearance of repair factors. However, our understanding of a role for deSUMOylation in this process is limited. Here we identify different mechanistic roles for deSUMOylation in homologous recombination (HR) and non-homologous enjoining (NHEJ) through the investigation of the deSUMOylase SENP2. We find regulated deSUMOylation of MDC1 prevents excessive SUMOylation and its RNF4-VCP mediated clearance from DSBs, thereby promoting NHEJ. In contrast we show HR is differentially sensitive to SUMO availability and SENP2 activity is needed to provide SUMO. SENP2 is amplified as part of the chromosome 3q amplification in many cancers. Increased SENP2 expression prolongs MDC1 foci retention and increases NHEJ and radioresistance. Collectively our data reveal that deSUMOylation differentially primes cells for responding to DSBs and demonstrates the ability of SENP2 to tune DSB repair responses.

PD-L1 upregulation is associated with activation of the DNA double-strand break repair pathway in patients with colitic cancer

2021 ◽  
Author(s):  
Naoya Ozawa ◽  
Takehiko Yokobori ◽  
Katsuya Osone ◽  
Chika Katayama ◽  
Kunihiko Suga ◽  
...  
Keyword(s):  
Immune Cell ◽  
Strand Break ◽  
Double Strand Break ◽  
Chronic Inflammatory Disease ◽  
Sporadic Colorectal Cancer ◽  
Cancer Tissue ◽  
Repair Pathway ◽  
Dsb Repair ◽  
Dna Double Strand Break ◽  
Colitic Cancer

Abstract Ulcerative colitis (UC) is a DNA damage-associated chronic inflammatory disease; the DNA double-strand break (DSB) repair pathway participates in UC-associated dysplasia/colitic cancer carcinogenesis. The DSB/interferon regulatory factor-1 (IRF-1) pathway can induce PD-L1 expression transcriptionally. However, the association of PD-L1/DSB/IRF-1 with sporadic colorectal cancer (SCRC), and UC-associated dysplasia/colitic cancer, remains elusive. Therefore, we investigated the significance of the PD-L1/DSB repair pathway using samples from 17 SCRC and 12 UC patients with rare UC-associated dysplasia/colitic cancer cases by immunohistochemical analysis. We compared PD-L1 expression between patients with SCRC and UC-associated dysplasia/colitic cancer and determined the association between PD-L1 and the CD8+ T-cell/DSB/IRF-1 axis in UC-associated dysplasia/colitic cancer. PD-L1 expression in UC and UC-associated dysplasia/colitic cancer was higher than in normal mucosa or SCRC, and in CD8-positive T lymphocytes in UC-associated dysplasia/colitic cancer than in SCRC. Moreover, PD-L1 upregulation was associated with γH2AX (DSB marker) and IRF-1 upregulation in UC-associated dysplasia/colitic cancer. IRF-1 upregulation was associated with γH2AX upregulation in UC-associated dysplasia/colitic cancer but not in SCRC. Multicolour immunofluorescence staining validated γH2AX/IRF-1/PD-L1 co-expression in colitic cancer tissue sections. Thus, immune cell-induced inflammation might activate the DSB/IRF-1 axis, potentially serving as the primary regulatory mechanism of PD-L1 expression in UC-associated carcinogenesis.

A Double Strand Break during Telophase Is Repaired with Homologous Recombination Despite the Absence of an Available Sister Chromatid

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2421-2421
Author(s):  
Amit Patel ◽  
Luis Alcaide Aragon
Keyword(s):  
Homologous Recombination ◽  
Sister Chromatid ◽  
Conflicts Of Interest ◽  
Strand Break ◽  
Double Strand Break ◽  
Restriction Enzyme Digestion ◽  
Dsb Repair ◽  
Sister Chromatids ◽  
Kinetics Of ◽  
In Cells

Abstract Background: Chromosomal breakage results from a DNA double strand break (DSB), and is repaired to maintain and restore genetic integrity, principally through two major pathways: homologous recombination (HR) and non-homologous end-joining (NHEJ). HR is initiated by nucleolytic resection of a DSB in the presence of cyclin-dependent kinase 1 (Cdk1) activity. DSB repair through HR is dependent on Rad52, and can be error-free when a sister chromatid is used as a template for repair. However, HR is mutagenic when any other template is used for repair. Loss of nucleotides adjacent to the DSB is a feature of repair through NHEJ. There is co-relation between Cdk1 activity and the presence of a sister chromatid. The research question was, in addition to Cdk1 activity is the presence of an intact sister chromatid a requirement to initiate DSB repair with the HR pathway. Methods: Cdk1 activity peaks during mitosis in the presence of an intact sister chromatid. To study DSB resection and repair in cells arrested in either mitotic metaphase or telophase when Cdk1-Clb2 was active, conditional alleles were constructed in a eukaryotic haploid budding yeast model of HR. The model permitted simultaneous induction of a single site-specific DSB in cells that were synchronised to a phase of the cell division cycle. Physical monitoring of the kinetics of DSB formation, nucleolytic resection of adjacent DNA, and DSB repair, was achieved by probing Southern membranes after restriction enzyme digestion of extracted genomic DNA from time courses. Results: Sister chromatids were segregated during telophase arrest induced by either Cdc14 or Cdc15 depletion. Metaphase arrest was achieved with Cdc20 depletion, either directly, or indirectly by activation of the spindle assembly checkpoint by inhibition of microtubule polymerisation. Sister chromatids were unsegregated and physically attached through cohesin during metaphase. The absence of an intact sister chromatid did not prevent DSB repair with the HR pathway during telophase. Nucleolytic resection was observed in the presence or absence of an intra-chromosomal homologous but non-identical DNA repair template. The DSB cut site did not become resistant to cycles of re-cleavage through loss of adjacent nucleotides. DSB repair by HR was dependent on Rad52. The kinetics of nucleolytic resection adjacent to the DSB, and repair by HR, were similar during telophase and metaphase. Conclusions: This is the first study to report the observation that the availability of the sister chromatid is not a requirement to promote DSB repair with the HR pathway during telophase. Initiation of HR occurs despite segregated sister chromatids, even in the absence of a non-identical homologous DNA donor template, with inherently mutagenic repair by HR. This unexpected discovery has important clinical implications to the pathogenesis of chromosomal translocations and oncogenesis, and tumour progression with repair of treatment-induced DSBs. Disclosures No relevant conflicts of interest to declare.

Impact of chromatin context on Cas9-induced DNA double-strand break repair pathway balance

2020 ◽  
Author(s):  
Ruben Schep ◽  
Eva K. Brinkman ◽  
Christ Leemans ◽  
Xabier Vergara ◽  
Ben Morris ◽  
...  
Keyword(s):  
Genome Editing ◽  
Strand Break ◽  
Double Strand Break ◽  
Repair Pathway ◽  
End Joining ◽  
Dsb Repair ◽  
Dna Double Strand Break ◽  
Repair Pathways ◽  
Non Homologous End Joining ◽  
The Impact

AbstractDNA double-strand break (DSB) repair is mediated by multiple pathways, including classical non-homologous end-joining pathway (NHEJ) and several homology-driven repair pathways. This is particularly important for Cas9-mediated genome editing, where the outcome critically depends on the pathway that repairs the break. It is thought that the local chromatin context affects the pathway choice, but the underlying principles are poorly understood. Using a newly developed multiplexed reporter assay in combination with Cas9 cutting, we systematically measured the relative activities of three DSB repair pathways as function of chromatin context in >1,000 genomic locations. This revealed that NHEJ is broadly biased towards euchromatin, while microhomology-mediated end-joining (MMEJ) is more efficient in specific heterochromatin contexts. In H3K27me3-marked heterochromatin, inhibition of the H3K27 methyltransferase EZH2 shifts the balance towards NHEJ. Single-strand templated repair (SSTR), often used for precise CRISPR editing, competes with MMEJ, and this competition is weakly associated with chromatin context. These results provide insight into the impact of chromatin on DSB repair pathway balance, and guidance for the design of Cas9-mediated genome editing experiments.

scholarly journals FKBP25 participates in DNA double-strand break repair

2020 ◽  
Vol 98 (1) ◽  
pp. 42-49 ◽  
Author(s):  
David Dilworth ◽  
Fade Gong ◽  
Kyle Miller ◽  
Christopher J. Nelson
Keyword(s):  
Homologous Recombination ◽  
Strand Break ◽  
Repair Pathway ◽  
Dna Double Strand Breaks ◽  
Dsb Repair ◽  
Peptide Bonds ◽  
Single Strand Annealing ◽  
Strand Annealing ◽  
Fk506 Binding Proteins

FK506-binding proteins (FKBPs) alter the conformation of proteins via cis–trans isomerization of prolyl-peptide bonds. While this activity can be demonstrated in vitro, the intractability of detecting prolyl isomerization events in cells has limited our understanding of the biological processes regulated by FKBPs. Here we report that FKBP25 is an active participant in the repair of DNA double-strand breaks (DSBs). FKBP25 influences DSB repair pathway choice by promoting homologous recombination (HR) and suppressing single-strand annealing (SSA). Consistent with this observation, cells depleted of FKBP25 form fewer Rad51 repair foci in response to etoposide and ionizing radiation, and they are reliant on the SSA repair factor Rad52 for viability. We find that FKBP25’s catalytic activity is required for promoting DNA repair, which is the first description of a biological function for this enzyme activity. Consistent with the importance of the FKBP catalytic site in HR, rapamycin treatment also impairs homologous recombination, and this effect is at least in part independent of mTor. Taken together these results identify FKBP25 as a component of the DNA DSB repair pathway.

scholarly journals Analysis of the Xenopus Werner syndrome protein in DNA double-strand break repair

2005 ◽  
Vol 171 (2) ◽  
pp. 217-227 ◽  
Author(s):  
Hong Yan ◽  
Jill McCane ◽  
Thomas Toczylowski ◽  
Chinyi Chen
Keyword(s):  
Werner Syndrome ◽  
Strand Break ◽  
Double Strand Break ◽  
Single Strand ◽  
Repair Pathway ◽  
Dsb Repair ◽  
Single Strand Annealing ◽  
Werner Syndrome Protein ◽  
Strand Annealing ◽  
Syndrome Protein

Werner syndrome is associated with premature aging and increased risk of cancer. Werner syndrome protein (WRN) is a RecQ-type DNA helicase, which seems to participate in DNA replication, double-strand break (DSB) repair, and telomere maintenance; however, its exact function remains elusive. Using Xenopus egg extracts as the model system, we found that Xenopus WRN (xWRN) is recruited to discrete foci upon induction of DSBs. Depletion of xWRN has no significant effect on nonhomologous end-joining of DSB ends, but it causes a significant reduction in the homology-dependent single-strand annealing DSB repair pathway. These results provide the first direct biochemical evidence that links WRN to a specific DSB repair pathway. The assay for single-strand annealing that was developed in this study also provides a powerful biochemical system for mechanistic analysis of homology-dependent DSB repair.

A unique pathway of double-strand break repair operates in tandemly repeated genes

1991 ◽  
Vol 11 (3) ◽  
pp. 1222-1231 ◽  
Author(s):  
B A Ozenberger ◽  
G S Roeder
Keyword(s):  
Gene Product ◽  
Gene Array ◽  
Strand Break ◽  
Double Strand Break ◽  
Dsb Repair ◽  
Yeast Saccharomyces Cerevisiae ◽  
Spontaneous Recombination ◽  
Genome Recombination ◽  
Repeat Units ◽  
Almost All

The RAD52 gene product of the yeast Saccharomyces cerevisiae is required for most spontaneous recombination and almost all double-strand break (DSB) repair. In contrast to recombination elsewhere in the genome, recombination in the ribosomal DNA (rDNA) array is RAD52 independent. To determine the fate of a DSB in the rDNA gene array, a cut site for the HO endonuclease was inserted into the rDNA in a strain containing an inducible HO gene. DSBs were efficiently repaired at this site, even in the absence of the RAD52 gene product. Efficient RAD52-independent DSB repair was also observed at another tandem gene array, CUP1, consisting of 18 repeat units. However, in a smaller CUP1 array, consisting of only three units, most DSBs (ca. 80%) were not repaired and resulted in cell death. All RAD52-independent DSB repair events examined resulted in the loss of one or more repeat units. We propose a model for DSB repair in repeated sequences involving the generation of single-stranded tails followed by reannealing.

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