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

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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Genome Instability and Pressure on Non-Homologous end Joining drives Chemotherapy Resistance via a DNA Repair Crisis Switch in Triple Negative Breast Cancer.

10.21203/rs.3.rs-103661/v1 ◽

2020 ◽

Author(s):

Adrian Wiegmans ◽

Ambber Ward ◽

Ekaterina Ivanova ◽

Pascal H G Duijf ◽

Romy VanOosterhout ◽

...

Keyword(s):

Breast Cancer ◽

Dna Repair ◽

Homologous Recombination ◽

Triple Negative Breast Cancer ◽

Triple Negative ◽

Genome Instability ◽

Chemotherapy Resistance ◽

End Joining ◽

Repair Pathways ◽

Non Homologous End Joining

Abstract Background: Chemotherapy intensifies pressure on the DNA repair pathways that can lead to deregulation. There is an urgent clinical need to be able to track the emergence of chemotherapy resistance and tailor patient staging appropriately. This is especially evident in the triple negative breast cancer (TNBC) subtype, of which standard of care is chemotherapy with tumours displaying high levels of inherent genome instability. TNBC has an overall poor prognosis for survival. There have been numerous studies into single agent chemoresistance but to date no study has elucidated in detail the roles of the key DNA repair components in resistance associated with the frontline clinical combination of anthracyclines and taxanes together. Methods: In this study, we hypothesized that the emergence of chemotherapy resistance is driven by changes in functional signaling in the DNA repair pathways. We identified the importance of the DNA repair pathways in chemoresistant clinical samples and characterized the emergence of chemoresistance in TNBC cell lines. We utilized classical DNA repair assays and specific targeting of key DNA repair proteins to elucidate a new mechanism for adaptation to the combination of doxorubicin and docetaxel. Results: We identified that consistent pressure on the non-homologous end joining pathway in the presence of genome instability causes failure of the key kinase DNA-PK, loss of p53 and compensation by p73. In-turn a switch to reliance on the homologous recombination pathway and RAD51 recombinase occurs to repair residual double strand DNA breaks. Conclusions: We demonstrate that RAD51 is an actionable target for resensitization to chemotherapy in resistant cells with a matched gene expression profile of resistance highlighted by homologous recombination.

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Genome instability and pressure on non-homologous end joining drives chemotherapy resistance via a DNA repair crisis switch in triple negative breast cancer

NAR Cancer ◽

10.1093/narcan/zcab022 ◽

2021 ◽

Vol 3 (2) ◽

Author(s):

Adrian P Wiegmans ◽

Ambber Ward ◽

Ekaterina Ivanova ◽

Pascal H G Duijf ◽

Mark N Adams ◽

...

Keyword(s):

Breast Cancer ◽

Dna Repair ◽

Homologous Recombination ◽

Triple Negative Breast Cancer ◽

Triple Negative ◽

Genome Instability ◽

Chemotherapy Resistance ◽

End Joining ◽

Repair Pathways ◽

Non Homologous End Joining

Abstract Chemotherapy is used as a standard-of-care against cancers that display high levels of inherent genome instability. Chemotherapy induces DNA damage and intensifies pressure on the DNA repair pathways that can lead to deregulation. There is an urgent clinical need to be able to track the emergence of DNA repair driven chemotherapy resistance and tailor patient staging appropriately. There have been numerous studies into chemoresistance but to date no study has elucidated in detail the roles of the key DNA repair components in resistance associated with the frontline clinical combination of anthracyclines and taxanes together. In this study, we hypothesized that the emergence of chemotherapy resistance in triple negative breast cancer was driven by changes in functional signaling in the DNA repair pathways. We identified that consistent pressure on the non-homologous end joining pathway in the presence of genome instability causes failure of the key kinase DNA-PK, loss of p53 and compensation by p73. In-turn a switch to reliance on the homologous recombination pathway and RAD51 recombinase occurred to repair residual double strand DNA breaks. Further we demonstrate that RAD51 is an actionable target for resensitization to chemotherapy in resistant cells with a matched gene expression profile of resistance highlighted by homologous recombination in clinical samples.

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Different DNA repair pathways are required following excision and integration of the DNA cut & paste transposon piggyBat in Saccharomyces cerevisiae.

10.1101/015289 ◽

2015 ◽

Cited By ~ 1

Author(s):

Weifeng She ◽

Courtney Busch Cambouris ◽

Nancy L. Craig

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Repair ◽

Single Gene ◽

Donor Site ◽

Insertion Site ◽

Strand Break ◽

Host Factors ◽

A Genome ◽

Genes Encoding ◽

Repair Pathways

The movement of transposable elements from place to place in a genome requires both element-encoded and host-encoded factors. In DNA cut & paste transposition, the element-encoded transposase performs the DNA breakage and joining reactions that excise the element from the donor site and integrate it into the new insertion site. Host factors can influence many aspects of transposition. Notably, host DNA repair factors mediate the regeneration of intact duplex DNA necessary after transposase action by repairing the double strand break in the broken donor backbone, from which the transposon has excised, and repairing the single strand gaps that flank the newly inserted transposon. We have exploited the ability of the mammalian transposon piggyBat, a member of the piggyBac superfamily, to transpose in Saccharomyces cerevisiae and used the yeast single gene deletion collection to screen for genes encoding host factors involved in piggyBat transposition. piggyBac transposition is distinguished by the fact that piggyBac elements insert into TTAA target sites and also that the donor backbone is restored to its pre-transposon sequence after transposon excision, that is, excision is precise. We have found that repair of the broken donor backbone requires the non-homologous end-joining repair pathway (NHEJ). By contrast, NHEJ is not required for DNA repair at the new insertion site. Thus multiple DNA repair pathways are required for piggyBac transposition.

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PARP Inhibitors as a Therapeutic Agent for Homologous Recombination Deficiency in Breast Cancers

Journal of Clinical Medicine ◽

10.3390/jcm8040435 ◽

2019 ◽

Vol 8 (4) ◽

pp. 435 ◽

Cited By ~ 29

Author(s):

Man Keung ◽

Yanyuan Wu ◽

Jaydutt Vadgama

Keyword(s):

Dna Repair ◽

Homologous Recombination ◽

Cancer Cells ◽

Anticancer Agents ◽

Excision Repair ◽

Parp Inhibitor ◽

Predictive Biomarkers ◽

Parp Inhibitors ◽

Homologous Recombination Deficiency ◽

Repair Pathways

Poly (ADP-ribose) polymerases (PARPs) play an important role in various cellular processes, such as replication, recombination, chromatin remodeling, and DNA repair. Emphasizing PARP’s role in facilitating DNA repair, the PARP pathway has been a target for cancer researchers in developing compounds which selectively target cancer cells and increase sensitivity of cancer cells to other anticancer agents, but which also leave normal cells unaffected. Since certain tumors (BRCA1/2 mutants) have deficient homologous recombination repair pathways, they depend on PARP-mediated base excision repair for survival. Thus, inhibition of PARP is a promising strategy to selectively kill cancer cells by inactivating complementary DNA repair pathways. Although PARP inhibitor therapy has predominantly targeted BRCA-mutated cancers, this review also highlights the growing conversation around PARP inhibitor treatment for non-BRCA-mutant tumors, those which exhibit BRCAness and homologous recombination deficiency. We provide an update on the field’s progress by considering PARP inhibitor mechanisms, predictive biomarkers, and clinical trials of PARP inhibitors in development. Bringing light to these findings would provide a basis for expanding the use of PARP inhibitors beyond BRCA-mutant breast tumors.

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CRISPR-Cas9 genome editing in human cells works via the Fanconi Anemia pathway

10.1101/136028 ◽

2017 ◽

Cited By ~ 17

Author(s):

Chris D Richardson ◽

Katelynn R Kazane ◽

Sharon J Feng ◽

Nicholas L Bray ◽

Axel J Schäfer ◽

...

Keyword(s):

Dna Repair ◽

Genome Editing ◽

Fanconi Anemia ◽

High Efficiency ◽

Genetic Diseases ◽

Dermal Fibroblasts ◽

Human Cells ◽

Individual Gene ◽

Break Site ◽

Repair Pathways

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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NUCKS1 promotes RAD54 activity in homologous recombination DNA repair

The Journal of Cell Biology ◽

10.1083/jcb.201911049 ◽

2020 ◽

Vol 219 (10) ◽

Author(s):

David G. Maranon ◽

Neelam Sharma ◽

Yuxin Huang ◽

Platon Selemenakis ◽

Meiling Wang ◽

...

Keyword(s):

Dna Repair ◽

Homologous Recombination ◽

Human Cells ◽

Cyclin Dependent Kinase ◽

Loop Formation ◽

Multifunctional Protein ◽

Kinase Substrate ◽

Dna Damage Signaling ◽

Dna Damaging Agents

NUCKS1 (nuclear ubiquitous casein kinase and cyclin-dependent kinase substrate 1) is a chromatin-associated, vertebrate-specific, and multifunctional protein with a role in DNA damage signaling and repair. Previously, we have shown that NUCKS1 helps maintain homologous recombination (HR) DNA repair in human cells and functions as a tumor suppressor in mice. However, the mechanisms by which NUCKS1 positively impacts these processes had remained unclear. Here, we show that NUCKS1 physically and functionally interacts with the DNA motor protein RAD54. Upon exposure of human cells to DNA-damaging agents, NUCKS1 controls the resolution of RAD54 foci. In unperturbed cells, NUCKS1 prevents RAD54’s inappropriate engagement with RAD51AP1. In vitro, NUCKS1 stimulates the ATPase activity of RAD54 and the RAD51–RAD54-mediated strand invasion step during displacement loop formation. Taken together, our data demonstrate that the NUCKS1 protein is an important new regulator of the spatiotemporal events in HR.

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The Relevance of G-Quadruplexes for DNA Repair

International Journal of Molecular Sciences ◽

10.3390/ijms222212599 ◽

2021 ◽

Vol 22 (22) ◽

pp. 12599

Author(s):

Rebecca Linke ◽

Michaela Limmer ◽

Stefan Juranek ◽

Annkristin Heine ◽

Katrin Paeschke

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Genome Stability ◽

Genome Instability ◽

Genetic Disorders ◽

Telomere Maintenance ◽

Dna Structures ◽

G4 Dna ◽

G Quadruplex ◽

Repair Pathways

DNA molecules can adopt a variety of alternative structures. Among these structures are G-quadruplex DNA structures (G4s), which support cellular function by affecting transcription, translation, and telomere maintenance. These structures can also induce genome instability by stalling replication, increasing DNA damage, and recombination events. G-quadruplex-driven genome instability is connected to tumorigenesis and other genetic disorders. In recent years, the connection between genome stability, DNA repair and G4 formation was further underlined by the identification of multiple DNA repair proteins and ligands which bind and stabilize said G4 structures to block specific DNA repair pathways. The relevance of G4s for different DNA repair pathways is complex and depends on the repair pathway itself. G4 structures can induce DNA damage and block efficient DNA repair, but they can also support the activity and function of certain repair pathways. In this review, we highlight the roles and consequences of G4 DNA structures for DNA repair initiation, processing, and the efficiency of various DNA repair pathways.

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