scholarly journals Mechanisms of Double-Strand-Break Repair During Gene Targeting in Mammalian Cells

Genetics ◽  
1999 ◽  
Vol 151 (3) ◽  
pp. 1127-1141 ◽  
Author(s):  
Philip Ng ◽  
Mark D Baker
Keyword(s):  
Gene Targeting ◽  
Restriction Enzyme ◽  
Mammalian Cells ◽  
Repair Process ◽  
Strand Break ◽  
Double Strand Break ◽  
Patch Repair ◽  
Restriction Enzyme Site ◽  
Dsb Repair ◽  
Vector Borne

AbstractIn the present study, the mechanism of double-strand-break (DSB) repair during gene targeting at the chromosomal immunoglobulin μ-locus in a murine hybridoma was examined. The gene-targeting assay utilized specially designed insertion vectors genetically marked in the region of homology to the chromosomal μ-locus by six diagnostic restriction enzyme site markers. The restriction enzyme markers permitted the contribution of vector-borne and chromosomal μ-sequences in the recombinant product to be determined. The use of the insertion vectors in conjunction with a plating procedure in which individual integrative homologous recombination events were retained for analysis revealed several important features about the mammalian DSB repair process: The presence of the markers within the region of shared homology did not affect the efficiency of gene targeting.In the majority of recombinants, the vector-borne marker proximal to the DSB was absent, being replaced with the corresponding chromosomal restriction enzyme site. This result is consistent with either formation and repair of a vector-borne gap or an “end” bias in mismatch repair of heteroduplex DNA (hDNA) that favored the chromosomal sequence.Formation of hDNA was frequently associated with gene targeting and, in most cases, began ∼645 bp from the DSB and could encompass a distance of at least 1469 bp.The hDNA was efficiently repaired prior to DNA replication.The repair of adjacent mismatches in hDNA occurred predominantly on the same strand, suggesting the involvement of a long-patch repair mechanism.

scholarly journals Use of a Small Palindrome Genetic Marker to Investigate Mechanisms of Double-Strand-Break Repair in Mammalian Cells

Genetics ◽  
2000 ◽  
Vol 154 (3) ◽  
pp. 1281-1289 ◽  
Author(s):  
Julang Li ◽  
Mark D Baker
Keyword(s):  
Mismatch Repair ◽  
Mammalian Cells ◽  
Indirect Evidence ◽  
Strand Break ◽  
Double Strand Break ◽  
Double Strand Break Repair ◽  
Chromosomal Markers ◽  
Vector Borne ◽  
Break Repair ◽  
The One

Abstract We examined mechanisms of mammalian homologous recombination using a gene targeting assay in which the vector-borne region of homology to the chromosome bore small palindrome insertions that frequently escape mismatch repair when encompassed within heteroduplex DNA (hDNA). Our assay permitted the product(s) of each independent recombination event to be recovered for molecular analysis. The results revealed the following: (i) vector-borne double-strand break (DSB) processing usually did not yield a large double-strand gap (DSG); (ii) in 43% of the recombinants, the results were consistent with crossover at or near the DSB; and (iii) in the remaining recombinants, hDNA was an intermediate. The sectored (mixed) genotypes observed in 38% of the recombinants provided direct evidence for involvement of hDNA, while indirect evidence was obtained from the patterns of mismatch repair (MMR). Individual hDNA tracts were either long or short and asymmetric or symmetric on the one side of the DSB examined. Clonal analysis of the sectored recombinants revealed how vector-borne and chromosomal markers were linked in each strand of individual hDNA intermediates. As expected, vector-borne and chromosomal markers usually resided on opposite strands. However, in one recombinant, they were linked on the same strand. The results are discussed with particular reference to the double-strand-break repair (DSBR) model of recombination.

scholarly journals The histone modification reader ZCWPW1 links histone methylation to PRDM9-induced double-strand break repair

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Tao Huang ◽  
Shenli Yuan ◽  
Lei Gao ◽  
Mengjing Li ◽  
Xiaochen Yu ◽  
...  
Keyword(s):  
Histone Modification ◽  
Repair Process ◽  
Strand Break ◽  
Double Strand Break ◽  
Dependent Manner ◽  
Selection System ◽  
Dsb Repair ◽  
Knock Out ◽  
Recombination Hotspots ◽  
Knock Out Mice

The histone modification writer Prdm9 has been shown to deposit H3K4me3 and H3K36me3 at future double-strand break (DSB) sites during the very early stages of meiosis, but the reader of these marks remains unclear. Here, we demonstrate that Zcwpw1 is an H3K4me3 reader that is required for DSB repair and synapsis in mouse testes. We generated H3K4me3 reader-dead Zcwpw1 mutant mice and found that their spermatocytes were arrested at the pachytene-like stage, which phenocopies the Zcwpw1 knock–out mice. Based on various ChIP-seq and immunofluorescence analyses using several mutants, we found that Zcwpw1's occupancy on chromatin is strongly promoted by the histone-modification activity of PRDM9. Zcwpw1 localizes to DMC1-labelled hotspots in a largely Prdm9-dependent manner, where it facilitates completion of synapsis by mediating the DSB repair process. In sum, our study demonstrates the function of ZCWPW1 that acts as part of the selection system for epigenetics-based recombination hotspots in mammals.

Strand Invasion and DNA Synthesis From the Two 3′ Ends of a Double-Strand Break in Mammalian Cells

Genetics ◽  
2003 ◽  
Vol 163 (4) ◽  
pp. 1439-1447 ◽  
Author(s):  
Richard D McCulloch ◽  
Leah R Read ◽  
Mark D Baker
Keyword(s):  
Dna Synthesis ◽  
Mammalian Cells ◽  
Strand Break ◽  
Double Strand Break ◽  
Chromosomal Locus ◽  
Vector Integration ◽  
Vector Borne ◽  
Strand Invasion ◽  
Chromosomal Sequences ◽  
Targeting Vector

AbstractAnalysis of the crossover products recovered following transformation of mammalian cells with a sequence insertion (“ends-in”) gene-targeting vector revealed a novel class of recombinant. In this class of recombinants, a single vector copy has integrated into an ectopic genomic position, leaving the structure of the cognate chromosomal locus unaltered. Thus, in this respect, the recombinants resemble simple cases of random vector integration. However, the important difference is that the two paired 3′ vector ends have acquired endogenous, chromosomal sequences flanking both sides of the vector-borne double-strand break (DSB). In some cases, copying was extensive, extending >16 kb into nonhomologous flanking DNA. The results suggest that mammalian homologous recombination events can involve strand invasion and DNA synthesis by both 3′ ends of the DSB. These DNA interactions are a central, predicted feature of the DSBR model of recombination.

scholarly journals The histone modification reader ZCWPW1 links histone methylation to PRDM9-induced double strand break repair

2019 ◽  
Author(s):  
Tao Huang ◽  
Shenli Yuan ◽  
Lei Gao ◽  
Mengjing Li ◽  
Xiaochen Yu ◽  
...  
Keyword(s):  
Histone Modification ◽  
Repair Process ◽  
Strand Break ◽  
Double Strand Break ◽  
Dependent Manner ◽  
Selection System ◽  
Dsb Repair ◽  
Knock Out ◽  
Recombination Hotspots ◽  
Knock Out Mice

ABSTRACTThe histone modification writer PRDM9 has been shown to deposit H3K4me3 and H3K36me3 at future double-strand break (DSB) sites during the very early stages of meiosis, but the reader of these marks remains unclear. Here, we demonstrate that ZCWPW1 is an H3K4me3 reader that is required for DSB repair and synapsis in mouse testes. We generated H3K4me3 reader-dead ZCWPW1 mutant mice and found that their spermatocytes were arrested at the pachytene-like stage, which phenocopies the Zcwpw1 knock–out mice. Based on various ChIP-seq and immunofluorescence analyses using several mutants, we found that ZCWPW1’s occupancy on chromatin is strongly promoted by the histone-modification activity of PRDM9. ZCWPW1 localizes to DMC1-labelled hotspots in a largely PRDM9-dependent manner, where it facilitates completion of synapsis by mediating the DSB repair process. In sum, our study demonstrates the function of ZCWPW1 that acts as part of the selection system for epigenetics-based recombination hotspots in mammals.

scholarly journals Roles for the DNA-PK complex and 53BP1 in protecting ends from resection during DNA double-strand break repair

2020 ◽  
Vol 61 (5) ◽  
pp. 718-726 ◽  
Author(s):  
Atsushi Shibata ◽  
Penny A Jeggo
Keyword(s):  
Mammalian Cells ◽  
Binding Capacity ◽  
Susceptibility Gene ◽  
Strand Break ◽  
Double Strand Break ◽  
Breast Cancer Susceptibility Gene ◽  
Dsb Repair ◽  
Fast Kinetics ◽  
Dna Double Strand Break ◽  
Slow Kinetics

Abstract p53-binding protein 1 (53BP1) exerts distinct impacts in different situations involving DNA double-strand break (DSB) rejoining. Here we focus on how 53BP1 impacts upon the repair of ionising radiation-induced DSBs (IR-DSBs) and how it interfaces with Ku, the DNA end-binding component of canonical non-homologous end-joining (c-NHEJ), the major DSB repair pathway in mammalian cells. We delineate three forms of IR-DSB repair: resection-independent c-NHEJ, which rejoins most IR-DSBs with fast kinetics in G1 and G2, and Artemis and resection-dependent c-NHEJ and homologous recombination (HR), which repair IR-DSBs with slow kinetics in G1 and G2 phase, respectively. The fast component of DSB repair after X-ray exposure occurs via c-NHEJ with normal kinetics in the absence of 53BP1. Ku is highly abundant and has avid DNA end-binding capacity which restricts DNA end-resection and promotes resection-independent c-NHEJ at most IR-DSBs. Thus, 53BP1 is largely dispensable for resection-independent c-NHEJ. In contrast, 53BP1 is essential for the process of rejoining IR-DSBs with slow kinetics. This role requires 53BP1’s breast cancer susceptibility gene I (BRCA1) C-terminal (BRCT) 2 domain, persistent ataxia telangiectasia mutated (ATM) activation and potentially relaxation of compacted chromatin at heterochromatic-DSBs. In distinction, 53BP1 inhibits resection-dependent IR-DSB repair in G1 and G2, and this resection-inhibitory function can be counteracted by BRCA1. We discuss a model whereby most IR-DSBs are rapidly repaired by 53BP1-independent and resection-independent c-NHEJ due to the ability of Ku to inhibit resection, but, if delayed, then resection in the presence of Ku is triggered, the 53BP1 barrier comes into force and BRCA1 counteraction is required for resection.

scholarly journals Intron Homing With Limited Exon Homology: Illegitimate Double-Strand-Break Repair in Intron Acquisition by Phage T4

Genetics ◽  
1999 ◽  
Vol 153 (4) ◽  
pp. 1513-1523
Author(s):  
Monica M Parker ◽  
Maureen Belisle ◽  
Marlene Belfort
Keyword(s):  
Bacteriophage T4 ◽  
Repair Process ◽  
Strand Break ◽  
Double Strand Break ◽  
Dsb Repair ◽  
Intron Homing ◽  
Strand Invasion ◽  
Precipitous Decline ◽  
The Relationship ◽  
Genetic Assay

Abstract The td intron of bacteriophage T4 encodes a DNA endonuclease that initiates intron homing to cognate intronless alleles by a double-strand-break (DSB) repair process. A genetic assay was developed to analyze the relationship between exon homology and homing efficiency. Because models predict exonucleolytic processing of the cleaved recipient leading to homologous strand invasion of the donor allele, the assay was performed in wild-type and exonuclease-deficient (rnh or dexA) phage. Efficient homing was supported by exon lengths of 50 bp or greater, whereas more limited exon lengths led to a precipitous decline in homing levels. However, extensive homology in one exon still supported elevated homing levels when the other exon was completely absent. Analysis of these “one-sided” events revealed recombination junctions at ectopic sites of microhomology and implicated nucleolytic degradation in illegitimate DSB repair in T4. Interestingly, homing efficiency with extremely limiting exon homology was greatly elevated in phage deficient in the 3′-5′ exonuclease, DexA, suggesting that the length of 3′ tails is a major determinant of the efficiency of DSB repair. Together, these results suggest that illegitimate DSB repair may provide a means by which introns can invade ectopic sites.

scholarly journals The Molecular Basis of Multiple Vector Insertion by Gene Targeting in Mammalian Cells

Genetics ◽  
1999 ◽  
Vol 151 (3) ◽  
pp. 1143-1155 ◽  
Author(s):  
Philip Ng ◽  
Mark D Baker
Keyword(s):  
Gene Targeting ◽  
Molecular Basis ◽  
Mammalian Cells ◽  
Tandem Duplication ◽  
Single Copy ◽  
Strand Break ◽  
Vector Integration ◽  
Vector Borne ◽  
Enzyme Markers ◽  
Targeting Vector

Abstract Gene targeting using sequence insertion vectors generally results in integration of one copy of the targeting vector generating a tandem duplication of the cognate chromosomal region of homology. However, occasionally the target locus is found to contain >1 copy of the integrated vector. The mechanism by which the latter recombinants arise is not known. In the present study, we investigated the molecular basis by which multiple vectors become integrated at the chromosomal immunoglobulin μ locus in a murine hybridoma. To accomplish this, specially designed insertion vectors were constructed that included six diagnostic restriction enzyme markers in the Cμ region of homology to the target chromosomal μ locus. This enabled contributions by the vector-borne and chromosomal Cμ sequences at the recombinant locus to be ascertained. Targeted recombinants were isolated and analyzed to determine the number of vector copies integrated at the chromosomal immunoglobulin μ locus. Targeted recombinants identified as bearing >1 copy of the integrated vector resulted from a Cμ triplication formed by two vector copies in tandem. Examination of the fate of the Cμ region markers suggested that this class of recombinant was generated predominantly, if not exclusively, by two targeted vector integration events, each involving insertion of a single copy of the vector. Both vector insertion events into the chromosomal μ locus were consistent with the double-strand-break repair mechanism of homologous recombination. We interpret our results, taken together, to mean that a proportion of recipient cells is in a predetermined state that is amenable to targeted but not random vector integration.

scholarly journals Quantitative analysis shows that repair of Cas9-induced double-strand DNA breaks is slow and error-prone

2017 ◽  
Author(s):  
Eva K. Brinkman ◽  
Tao Chen ◽  
Marcel de Haas ◽  
Hanna A. Holland ◽  
Waseem Akhtar ◽  
...  
Keyword(s):  
Repair Process ◽  
Strand Break ◽  
Double Strand Break ◽  
Dna Breaks ◽  
End Joining ◽  
Dsb Repair ◽  
Double Strand Dna Breaks ◽  
Chemical Inhibition ◽  
Changes Over Time ◽  
Kinetics Of

SummaryThe RNA-guided DNA endonuclease Cas9 is a powerful tool for genome editing. Little is known about the kinetics and fidelity of the double-strand break (DSB) repair process that follows a Cas9 cutting event in living cells. Here, we developed a strategy to measure the kinetics of DSB repair for single loci in human cells. Quantitative modeling of repaired DNA in time series after Cas9 activation reveals a relatively slow repair rate (~6h). Furthermore, the double strand break is predominantly repaired in an error-prone fashion (at least 70%). Both classical and microhomology-mediated end-joining pathways are active and contribute to the repair in a stochastic manner. However, the balance between these two pathways changes over time and can be altered by chemical inhibition of DNAPKcs or additional ionizing radiation. Our strategy is generally applicable to study DSB repair kinetics and fidelity in single loci, and demonstrates that Cas9-induced DSBs are repaired in an unusual manner.

scholarly journals Rad51-Independent Interchromosomal Double-Strand Break Repair by Gene Conversion Requires Rad52 but Not Rad55, Rad57, or Dmc1

2007 ◽  
Vol 28 (3) ◽  
pp. 897-906 ◽  
Author(s):  
Thomas J. Pohl ◽  
Jac A. Nickoloff
Keyword(s):  
Gene Conversion ◽  
Strand Break ◽  
Double Strand Break ◽  
Single Strand ◽  
Homology Search ◽  
Wild Type ◽  
Dsb Repair ◽  
Single Strand Annealing ◽  
In The Wild ◽  
Break Induced Replication

ABSTRACT Homologous recombination (HR) is critical for DNA double-strand break (DSB) repair and genome stabilization. In yeast, HR is catalyzed by the Rad51 strand transferase and its “mediators,” including the Rad52 single-strand DNA-annealing protein, two Rad51 paralogs (Rad55 and Rad57), and Rad54. A Rad51 homolog, Dmc1, is important for meiotic HR. In wild-type cells, most DSB repair results in gene conversion, a conservative HR outcome. Because Rad51 plays a central role in the homology search and strand invasion steps, DSBs either are not repaired or are repaired by nonconservative single-strand annealing or break-induced replication mechanisms in rad51Δ mutants. Although DSB repair by gene conversion in the absence of Rad51 has been reported for ectopic HR events (e.g., inverted repeats or between plasmids), Rad51 has been thought to be essential for DSB repair by conservative interchromosomal (allelic) gene conversion. Here, we demonstrate that DSBs stimulate gene conversion between homologous chromosomes (allelic conversion) by >30-fold in a rad51Δ mutant. We show that Rad51-independent allelic conversion and break-induced replication occur independently of Rad55, Rad57, and Dmc1 but require Rad52. Unlike DSB-induced events, spontaneous allelic conversion was detected in both rad51Δ and rad52Δ mutants, but not in a rad51Δ rad52Δ double mutant. The frequencies of crossovers associated with DSB-induced gene conversion were similar in the wild type and the rad51Δ mutant, but discontinuous conversion tracts were fivefold more frequent and tract lengths were more widely distributed in the rad51Δ mutant, indicating that heteroduplex DNA has an altered structure, or is processed differently, in the absence of Rad51.

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