Mechanisms restraining break‐induced replication at two‐ended DNA double‐strand breaks

Pham, Nhung; Yan, Zhong; Yu, Yang; Afreen, Mosammat Faria; Malkova, Anna; Haber, James E.; Ira, Grzegorz

doi:10.15252/embj.2020104847

Cited by 50 publications

(55 citation statements)

References 98 publications

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“…The mechanism of CN-LOH post CRISPR-Cas9-mediated DSB is probably consistent with Break-induced replication (BIR) recently described in eukaryotic cells 31 . It involves extensive DNA synthesis from DSB to the telomere 32 contrary to classical gene conversion with only a short kilobase-scale patch of DNA synthesis in the neighboring of the DSB 33 . DNA synthesis is imprecise during BIR process.…”

Section: Discussionmentioning

confidence: 99%

CRISPR-Cas9 globin editing can induce megabase-scale copy-neutral losses of heterozygosity in hematopoietic cells

et al. 2021

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CRISPR-Cas9 is a promising technology for gene therapy. However, the ON-target genotoxicity of CRISPR-Cas9 nuclease due to DNA double-strand breaks has received little attention and is probably underestimated. Here we report that genome editing targeting globin genes induces megabase-scale losses of heterozygosity (LOH) from the globin CRISPR-Cas9 cut-site to the telomere (5.2 Mb). In established lines, CRISPR-Cas9 nuclease induces frequent terminal chromosome 11p truncations and rare copy-neutral LOH. In primary hematopoietic progenitor/stem cells, we detect 1.1% of clones (7/648) with acquired megabase LOH induced by CRISPR-Cas9. In-depth analysis by SNP-array reveals the presence of copy-neutral LOH. This leads to 11p15.5 partial uniparental disomy, comprising two Chr11p15.5 imprinting centers (H19/IGF2:IG-DMR/IC1 and KCNQ1OT1:TSS-DMR/IC2) and impacting H19 and IGF2 expression. While this genotoxicity is a safety concern for CRISPR clinical trials, it is also an opportunity to model copy-neutral-LOH for genetic diseases and cancers.

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Section: Discussionmentioning

confidence: 99%

CRISPR-Cas9 globin editing can induce megabase-scale copy-neutral losses of heterozygosity in hematopoietic cells

et al. 2021

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“…Break-induced replication [80] and DSB-independent replication template exchange [6,81] also require Pol δ. Although a mutation in a Cdc27/ Pol32/POLD3 subunit of Pol δ did not reduce GCRs in rad51Δ cells [11], Pol δ could be required for isochromosome formation in rad51Δ rad52Δ cells [82]. One of the future directions is to understand how homology-mediated GCRs occur independently of Rad52.…”

Section: Discussionmentioning

confidence: 99%

Fission yeast Rad8/HLTF facilitates Rad52-dependent chromosomal rearrangements through PCNA lysine 107 ubiquitination

Mongia

et al. 2021

PLoS Genet

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Gross chromosomal rearrangements (GCRs), including translocation, deletion, and inversion, can cause cell death and genetic diseases such as cancer in multicellular organisms. Rad51, a DNA strand exchange protein, suppresses GCRs by repairing spontaneous DNA damage through a conservative way of homologous recombination, gene conversion. On the other hand, Rad52 that catalyzes single-strand annealing (SSA) causes GCRs using homologous sequences. However, the detailed mechanism of Rad52-dependent GCRs remains unclear. Here, we provide genetic evidence that fission yeast Rad8/HLTF facilitates Rad52-dependent GCRs through the ubiquitination of lysine 107 (K107) of PCNA, a DNA sliding clamp. In rad51Δ cells, loss of Rad8 eliminated 75% of the isochromosomes resulting from centromere inverted repeat recombination, showing that Rad8 is essential for the formation of the majority of isochromosomes in rad51Δ cells. Rad8 HIRAN and RING finger mutations reduced GCRs, suggesting that Rad8 facilitates GCRs through 3’ DNA-end binding and ubiquitin ligase activity. Mms2 and Ubc4 but not Ubc13 ubiquitin-conjugating enzymes were required for GCRs. Consistent with this, mutating PCNA K107 rather than the well-studied PCNA K164 reduced GCRs. Rad8-dependent PCNA K107 ubiquitination facilitates Rad52-dependent GCRs, as PCNA K107R, rad8, and rad52 mutations epistatically reduced GCRs. In contrast to GCRs, PCNA K107R did not significantly change gene conversion rates, suggesting a specific role of PCNA K107 ubiquitination in GCRs. PCNA K107R enhanced temperature-sensitive growth defects of DNA ligase I cdc17-K42 mutant, implying that PCNA K107 ubiquitination occurs when Okazaki fragment maturation fails. Remarkably, K107 is located at the interface between PCNA subunits, and an interface mutation D150E bypassed the requirement of PCNA K107 and Rad8 ubiquitin ligase for GCRs. These data suggest that Rad8-dependent PCNA K107 ubiquitination facilitates Rad52-dependent GCRs by changing the PCNA clamp structure.

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“…Fragile site FS2 consists of an inverted pair of retrotransposons ( Lemoine et al 2005 ), thus while one broken end initiated BIR using the homologous chromosome III as a template, the other broken end initiated BIR using a repetitive element in a nonhomologous chromosome. Normally, BIR is restrained at a two-ended DSB by coordinating the simultaneous engagement of both ends of the break, through Rad52p or Rad59 annealing of the second broken end, and through synchronous resection of both broken ends and possibly tethering by the Mre11-Rad50-Xrs2 (MRX) complex ( Pham et al 2021 ). However, the simultaneous engagement of broken ends that limits BIR would be precluded by significant temporal separation in when the two ends are available for repair.…”

Section: Discussionmentioning

confidence: 99%

Noncanonical outcomes of break-induced replication produce complex, extremely long-tract gene conversion events in yeast

Stewart

Hillegass

Oberlitner

et al. 2021

G3 Genes|Genomes|Genetics

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Long-tract gene conversions (LTGC) can result from the repair of collapsed replication forks, and several mechanisms have been proposed to explain how the repair process produces this outcome. We studied LTGC events produced from repair collapsed forks at yeast fragile site FS2. Our analysis included chromosome sizing by contour-clamped homogeneous electric field (CHEF) electrophoresis, next-generation whole genome sequencing, and Sanger sequencing across repair event junctions. We compared the sequence and structure of LTGC events in our cells to the expected qualities of LTGC events generated by proposed mechanisms. Our evidence indicates that some LTGC events arise from half-crossover during BIR, some LTGC events arise from gap repair, and some LTGC events can be explained by either gap repair or “late” template switch during BIR. Also based on our data, we propose that models of collapsed replication forks be revised to show not a one-end double-strand break (DSB), but rather a two-end DSB in which the ends are separated in time and subject to gap repair.

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Mechanisms restraining break‐induced replication at two‐ended DNA double‐strand breaks

Cited by 50 publications

References 98 publications

CRISPR-Cas9 globin editing can induce megabase-scale copy-neutral losses of heterozygosity in hematopoietic cells

CRISPR-Cas9 globin editing can induce megabase-scale copy-neutral losses of heterozygosity in hematopoietic cells

Fission yeast Rad8/HLTF facilitates Rad52-dependent chromosomal rearrangements through PCNA lysine 107 ubiquitination

Noncanonical outcomes of break-induced replication produce complex, extremely long-tract gene conversion events in yeast

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