BRCA1 and S phase DNA repair pathways restrict LINE-1 retrotransposition in human cells

AbstractCRISPR-Cas9 genome editing creates targeted double strand breaks (DSBs) in eukaryotic cells that are processed by cellular DNA repair pathways. Co-administration of single stranded oligonucleotide donor DNA (ssODN) during editing can result in high-efficiency (>20%) incorporation of ssODN sequences into the break site. This process is commonly referred to as homology directed repair (HDR) and here referred to as single stranded template repair (SSTR) to distinguish it from repair using a double stranded DNA donor (dsDonor). The high efficacy of SSTR makes it a promising avenue for the treatment of genetic diseases1,2, but the genetic basis of SSTR editing is still unclear, leaving its use a mostly empiric process. To determine the pathways underlying SSTR in human cells, we developed a coupled knockdown-editing screening system capable of interrogating multiple editing outcomes in the context of thousands of individual gene knockdowns. Unexpectedly, we found that SSTR requires multiple components of the Fanconi Anemia (FA) repair pathway, but does not require Rad51-mediated homologous recombination, distinguishing SSTR from repair using dsDonors. Knockdown of FA genes impacts SSTR without altering break repair by non-homologous end joining (NHEJ) in multiple human cell lines and in neonatal dermal fibroblasts. Our results establish an unanticipated and central role for the FA pathway in templated repair from single stranded DNA by human cells. Therapeutic genome editing has been proposed to treat genetic disorders caused by deficiencies in DNA repair, including Fanconi Anemia. Our data imply that patient genotype and/or transcriptome profoundly impact the effectiveness of gene editing treatments and that adjuvant treatments to bias cells towards FA repair pathways could have considerable therapeutic value.

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New Insight on Entangled DNA Repair Pathways: Stable Silenced Human Cells for Unraveling the DDR Jigsaw

DNA Repair - On the Pathways to Fixing DNA Damage and Errors ◽

10.5772/23681 ◽

2011 ◽

Author(s):

Biard Denis

Keyword(s):

Dna Repair ◽

Human Cells ◽

Repair Pathways

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Differential micronucleus frequency in isogenic human cells deficient in DNA repair pathways is a valuable indicator for evaluating genotoxic agents and their genotoxic mechanisms

Environmental and Molecular Mutagenesis ◽

10.1002/em.22201 ◽

2018 ◽

Vol 59 (6) ◽

pp. 529-538 ◽

Cited By ~ 5

Author(s):

Liton Kumar Saha ◽

Sujin Kim ◽

Habyeong Kang ◽

Salma Akter ◽

Kyungho Choi ◽

...

Keyword(s):

Dna Repair ◽

Human Cells ◽

Micronucleus Frequency ◽

Genotoxic Agents ◽

Repair Pathways

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p53 Activation by Cr(VI): A Transcriptionally Limited Response Induced by ATR Kinase in S-Phase

Toxicological Sciences ◽

10.1093/toxsci/kfz178 ◽

2019 ◽

Vol 172 (1) ◽

pp. 11-22 ◽

Cited By ~ 1

Author(s):

Michal W Luczak ◽

Casey Krawic ◽

Anatoly Zhitkovich

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Target Genes ◽

Cultured Cells ◽

Excision Repair ◽

S Phase ◽

Human Cells ◽

P53 Activation ◽

Atr Kinase ◽

Primary Human Cells

Abstract Cellular reduction of carcinogenic chromium(VI) causes several forms of Cr-DNA damage with different genotoxic properties. Chromate-treated cultured cells have shown a strong proapoptotic activity of the DNA damage-sensitive transcription factor p53. However, induction of p53 transcriptional targets by Cr(VI) in rodent lungs was weak or undetectable. We examined Cr(VI) effects on the p53 pathway in human cells with restored levels of ascorbate that acts as a principal reducer of Cr(VI) in vivo but is nearly absent in standard cell cultures. Ascorbate-restored H460 and primary human cells treated with Cr(VI) contained higher levels of p53 and its Ser15 phosphorylation, which were induced by ATR kinase. Cr(VI)-stimulated p53 phosphorylation occurred in S-phase by a diffusible pool of ATR that was separate from the chromatin-bound pool targeting DNA repair substrates at the sites of toxic mismatch repair (MMR) of Cr-DNA adducts. Even when more abundantly present than after exposure to the radiomimetic bleomycin, Cr(VI)-stabilized p53 showed a much more limited activation of its target genes in two types of primary human cells. No increases in mRNA were found for nucleotide excision repair factors and a majority of proapoptotic genes. A weak transcription activity of Cr(VI)-upregulated p53 was associated with its low lysine acetylation in the regulatory C-terminal domain, resulting from the inability of Cr(VI) to activate ATM in ascorbate-restored cells. Thus, p53 activation by ascorbate-metabolized Cr(VI) represents a limited genome-protective response that is defective in upregulation of DNA repair genes and proapoptotic transcripts for elimination of damaged cells.

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Preserving genome integrity in human cells via DNA double-strand break repair

Molecular Biology of the Cell ◽

10.1091/mbc.e18-10-0668 ◽

2020 ◽

Vol 31 (9) ◽

pp. 859-865 ◽

Cited By ~ 1

Author(s):

Ryan B. Jensen ◽

Eli Rothenberg

Keyword(s):

Dna Repair ◽

Strand Break ◽

Double Strand Break ◽

Human Cells ◽

Cell Cycle Checkpoints ◽

Human Diseases ◽

Genome Integrity ◽

Dsb Repair ◽

Dna Double Strand Break ◽

Repair Pathways

The efficient maintenance of genome integrity in the face of cellular stress is vital to protect against human diseases such as cancer. DNA replication, chromatin dynamics, cellular signaling, nuclear architecture, cell cycle checkpoints, and other cellular activities contribute to the delicate spatiotemporal control that cells utilize to regulate and maintain genome stability. This perspective will highlight DNA double-strand break (DSB) repair pathways in human cells, how DNA repair failures can lead to human disease, and how PARP inhibitors have emerged as a novel clinical therapy to treat homologous recombination-deficient tumors. We briefly discuss how failures in DNA repair produce a permissive genetic environment in which preneoplastic cells evolve to reach their full tumorigenic potential. Finally, we conclude that an in-depth understanding of DNA DSB repair pathways in human cells will lead to novel therapeutic strategies to treat cancer and potentially other human diseases.

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BRCA1 Mediated Homologous Recombination and S Phase DNA Repair Pathways Restrict LINE-1 Retrotransposition in Human Cells

10.1101/701458 ◽

2019 ◽

Author(s):

Xiaoji Sun ◽

Paolo Mita ◽

David J. Kahler ◽

Donghui Li ◽

Aleksandra Wudzinska ◽

...

Keyword(s):

Dna Repair ◽

Homologous Recombination ◽

Potential Role ◽

Genome Instability ◽

Protein Translation ◽

Human Cells ◽

Host Factors ◽

Factors Affecting ◽

A Genome ◽

Repair Pathways

AbstractLong interspersed element-1 (LINE-1 or L1) is the only autonomous retrotransposon active in human cells. L1s DNA makes about 17% of the human genome and retrotransposition of a few active L1 copies has been detected in various tumors, underscoring the potential role of L1 in mediating or increasing genome instability during tumorigenic development. Different host factors have been shown to influence L1 mobility through several mechanisms. However, systematic analyses of host factors affecting L1 retrotransposition are limited. Here, we developed a high-throughput microscopy-based retrotransposition assay and coupled it to a genome-wide siRNA knockdown screen to study the cellular regulators of L1 retrotransposition in human cells. We showed that L1 insertion frequency was stimulated by knockdown of Double-Stranded Break (DSB) repair factors that are active in the S/G2 phase of the cell cycle including Homologous Recombination (HR), Fanconi Anemia (FA) and, to a less extent, microhomology-mediated end-joining (MMEJ) factors. In particular, we show that BRCA1, an E3 ubiquitin ligase with a key role in several DNA repair pathways, plays multiple roles in regulating L1; BRCA1 knockdown directly affects L1 retrotransposition frequency and structure and also plays a role in controlling L1 ORF2 protein translation through L1 mRNA binding. These results suggest the existence of a “battle” between HR factors and L1 retrotransposons, revealing a potential role for L1 in development of tumors characterized by BRCA1 and HR repair deficiencies.

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The HMCES DNA-protein cross-link functions as a constitutive DNA repair intermediate

10.1101/2021.07.29.454365 ◽

2021 ◽

Author(s):

Daniel R Semlow ◽

Victoria A MacKrell ◽

Johannes Walter

Keyword(s):

Dna Repair ◽

Strand Break ◽

S Phase ◽

Cross Link ◽

Link Functions ◽

Cross Links ◽

Ap Sites ◽

Repair Pathways ◽

Break Formation ◽

Ap Site

The HMCES protein forms a covalent DNA-protein cross-link (DPC) with abasic (AP) sites in ssDNA, and the resulting HMCES-DPC is thought to suppress double-strand break formation in S phase. However, the dynamics of HMCES cross-linking and whether any DNA repair pathways normally include an HMCES-DPC intermediate remain unknown. Here, we show that an HMCES-DPC forms efficiently on the AP site generated during replication-coupled DNA interstrand cross-link (ICL) repair. We use this system to show that HMCES cross-links form on DNA after the replicative CMG helicase has passed over the AP site, and that HMCES is subsequently removed by the SPRTN protease. The HMCES-DPC suppresses DSB formation, slows translesion synthesis (TLS) past the AP site, and introduces a bias for insertion of deoxyguanosine opposite the AP site. These data show that HMCES-DPCs can form as constitutive intermediates in replication-coupled repair, and they suggest a general model of how HMCES protects AP sites during DNA replication.

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