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

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.

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

eLife ◽

10.7554/elife.53459 ◽

2020 ◽

Vol 9 ◽

Cited By ~ 4

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.

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

Genetics ◽

10.1093/genetics/151.3.1127 ◽

1999 ◽

Vol 151 (3) ◽

pp. 1127-1141 ◽

Cited By ~ 8

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.

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

10.1101/836023 ◽

2019 ◽

Cited By ~ 1

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.

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Coordination of DNA Ends During Double-Strand-Break Repair in Bacteriophage T4

Genetics ◽

10.1093/genetics/162.3.1019 ◽

2002 ◽

Vol 162 (3) ◽

pp. 1019-1030

Author(s):

Bradley A Stohr ◽

Kenneth N Kreuzer

Keyword(s):

Bacteriophage T4 ◽

Strand Break ◽

Double Strand Break ◽

Double Strand Break Repair ◽

Recombinational Repair ◽

Repair Template ◽

Strand Invasion ◽

Break Repair ◽

Template Dna ◽

Dna Ends

Abstract The extensive chromosome replication (ECR) model of double-strand-break repair (DSBR) proposes that each end of a double-strand break (DSB) is repaired independently by initiating extensive semiconservative DNA replication after strand invasion into homologous template DNA. In contrast, several other DSBR models propose that the two ends of a break are repaired in a coordinated manner using a single repair template with only limited DNA synthesis. We have developed plasmid and chromosomal recombinational repair assays to assess coordination of the broken ends during DSBR in bacteriophage T4. Results from the plasmid assay demonstrate that the two ends of a DSB can be repaired independently using homologous regions on two different plasmids and that extensive replication is triggered in the process. These findings are consistent with the ECR model of DSBR. However, results from the chromosomal assay imply that the two ends of a DSB utilize the same homologous repair template even when many potential templates are present, suggesting coordination of the broken ends during chromosomal repair. This result is consistent with several coordinated models of DSBR, including a modified version of the ECR model.

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

10.1101/142802 ◽

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.

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

Molecular and Cellular Biology ◽

10.1128/mcb.00524-07 ◽

2007 ◽

Vol 28 (3) ◽

pp. 897-906 ◽

Cited By ~ 21

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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Watching a double strand break repair polymerase insert a pro-mutagenic oxidized nucleotide

Nature Communications ◽

10.1038/s41467-021-21354-6 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Joonas A. Jamsen ◽

Akira Sassa ◽

David D. Shock ◽

William A. Beard ◽

Samuel H. Wilson

Keyword(s):

Active Site ◽

Dna Polymerases ◽

Time Lapse ◽

Strand Break ◽

Double Strand Break ◽

Double Strand Break Repair ◽

Dsb Repair ◽

X Ray Crystallography ◽

Polymerase Fidelity ◽

Break Repair

AbstractOxidized dGTP (8-oxo-7,8-dihydro-2´-deoxyguanosine triphosphate, 8-oxodGTP) insertion by DNA polymerases strongly promotes cancer and human disease. How DNA polymerases discriminate against oxidized and undamaged nucleotides, especially in error-prone double strand break (DSB) repair, is poorly understood. High-resolution time-lapse X-ray crystallography snapshots of DSB repair polymerase μ undergoing DNA synthesis reveal that a third active site metal promotes insertion of oxidized and undamaged dGTP in the canonical anti-conformation opposite template cytosine. The product metal bridged O8 with product oxygens, and was not observed in the syn-conformation opposite template adenine (At). Rotation of At into the syn-conformation enabled undamaged dGTP misinsertion. Exploiting metal and substrate dynamics in a rigid active site allows 8-oxodGTP to circumvent polymerase fidelity safeguards to promote pro-mutagenic double strand break repair.

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Repairing a double–strand chromosome break by homologous recombination: revisiting Robin Holliday's model

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2003.1367 ◽

2004 ◽

Vol 359 (1441) ◽

pp. 79-86 ◽

Cited By ~ 53

Author(s):

James E. Haber ◽

Gregorz Ira ◽

Anna Malkova ◽

Neal Sugawara

Keyword(s):

Homologous Recombination ◽

Repair Process ◽

Strand Break ◽

Holliday Junctions ◽

Chromosome Break ◽

Invasion Mechanism ◽

Strand Invasion ◽

Strand Annealing ◽

Break Induced Replication ◽

Mutant Cells

Since the pioneering model for homologous recombination proposed by Robin Holliday in 1964, there has been great progress in understanding how recombination occurs at a molecular level. In the budding yeast Saccharomyces cerevisiae , one can follow recombination by physically monitoring DNA after the synchronous induction of a double–strand break (DSB) in both wild–type and mutant cells. A particularly well–studied system has been the switching of yeast mating–type ( MAT ) genes, where a DSB can be induced synchronously by expression of the site–specific HO endonuclease. Similar studies can be performed in meiotic cells, where DSBs are created by the Spo11 nuclease. There appear to be at least two competing mechanisms of homologous recombination: a synthesis–dependent strand annealing pathway leading to noncrossovers and a two–end strand invasion mechanism leading to formation and resolution of Holliday junctions (HJs), leading to crossovers. The establishment of a modified replication fork during DSB repair links gene conversion to another important repair process, break–induced replication. Despite recent revelations, almost 40 years after Holliday's model was published, the essential ideas he proposed of strand invasion and heteroduplex DNA formation, the formation and resolution of HJs, and mismatch repair, remain the basis of our thinking.

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

Molecular and Cellular Biology ◽

10.1128/mcb.11.3.1222 ◽

1991 ◽

Vol 11 (3) ◽

pp. 1222-1231 ◽

Cited By ~ 85

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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PARP-1–dependent recruitment of cold-inducible RNA-binding protein promotes double-strand break repair and genome stability

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1713912115 ◽

2018 ◽

Vol 115 (8) ◽

pp. E1759-E1768 ◽

Cited By ~ 14

Author(s):

Jung-Kuei Chen ◽

Wen-Ling Lin ◽

Zhang Chen ◽

Hung-wen Liu

Keyword(s):

Dna Damage ◽

Genome Stability ◽

Rna Binding ◽

Binding Protein ◽

Rna Binding Protein ◽

Strand Break ◽

Double Strand Break ◽

Dsb Repair ◽

Active Involvement ◽

Parp 1

Maintenance of genome integrity is critical for both faithful propagation of genetic information and prevention of mutagenesis induced by various DNA damage events. Here we report cold-inducible RNA-binding protein (CIRBP) as a newly identified key regulator in DNA double-strand break (DSB) repair. On DNA damage, CIRBP temporarily accumulates at the damaged regions and is poly(ADP ribosyl)ated by poly(ADP ribose) polymerase-1 (PARP-1). Its dissociation from the sites of damage may depend on its phosphorylation status as mediated by phosphatidylinositol 3-kinase-related kinases. In the absence of CIRBP, cells showed reduced γH2AX, Rad51, and 53BP1 foci formation. Moreover, CIRBP-depleted cells exhibited impaired homologous recombination, impaired nonhomologous end-joining, increased micronuclei formation, and higher sensitivity to gamma irradiation, demonstrating the active involvement of CIRBP in DSB repair. Furthermore, CIRBP depleted cells exhibited defects in DNA damage-induced chromatin association of the MRN complex (Mre11, Rad50, and NBS1) and ATM kinase. CIRBP depletion also reduced phosphorylation of a variety of ATM substrate proteins and thus impaired the DNA damage response. Taken together, these results reveal a previously unrecognized role for CIRBP in DSB repair.

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