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Journal ArticleDOI: 10.1016/J.CELL.2021.01.048

RNA polymerase III is required for the repair of DNA double-strand breaks by homologous recombination.

04 Mar 2021-Cell (Elsevier)-Vol. 184, Iss: 5
Abstract: Summary End resection in homologous recombination (HR) and HR-mediated repair of DNA double-strand breaks (DSBs) removes several kilobases from 5′ strands of DSBs, but 3′ strands are exempted from degradation. The mechanism by which the 3′ overhangs are protected has not been determined. Here, we established that the protection of 3′ overhangs is achieved through the transient formation of RNA-DNA hybrids. The DNA strand in the hybrids is the 3′ ssDNA overhang, while the RNA strand is newly synthesized. RNA polymerase III (RNAPIII) is responsible for synthesizing the RNA strand. Furthermore, RNAPIII is actively recruited to DSBs by the MRN complex. CtIP and MRN nuclease activity is required for initiating the RNAPIII-mediated RNA synthesis at DSBs. A reduced level of RNAPIII suppressed HR, and genetic loss > 30 bp increased at DSBs. Thus, RNAPIII is an essential HR factor, and the RNA-DNA hybrid is an essential repair intermediate for protecting the 3′ overhangs in DSB repair.

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Topics: MRN complex (59%), DNA (53%), Homologous recombination (53%) ... show more
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28 results found


Open accessJournal ArticleDOI: 10.1371/JOURNAL.PGEN.1009459
Xue Cheng1, Valérie Côté1, Jacques Côté1Institutions (1)
06 Jul 2021-PLOS Genetics
Abstract: Chromatin modifying complexes play important yet not fully defined roles in DNA repair processes. The essential NuA4 histone acetyltransferase (HAT) complex is recruited to double-strand break (DSB) sites and spreads along with DNA end resection. As predicted, NuA4 acetylates surrounding nucleosomes upon DSB induction and defects in its activity correlate with altered DNA end resection and Rad51 recombinase recruitment. Importantly, we show that NuA4 is also recruited to the donor sequence during recombination along with increased H4 acetylation, indicating a direct role during strand invasion/D-loop formation after resection. We found that NuA4 cooperates locally with another HAT, the SAGA complex, during DSB repair as their combined action is essential for DNA end resection to occur. This cooperation of NuA4 and SAGA is required for recruitment of ATP-dependent chromatin remodelers, targeted acetylation of repair factors and homologous recombination. Our work reveals a multifaceted and conserved cooperation mechanism between acetyltransferase complexes to allow repair of DNA breaks by homologous recombination.

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Topics: DNA repair (61%), Strand invasion (58%), Homologous recombination (58%) ... show more

4 Citations


Open accessJournal ArticleDOI: 10.3389/FMOLB.2021.668821
Abstract: DNA double-strand breaks (DSBs) are highly cytotoxic DNA lesions. To protect genomic stability and ensure cell homeostasis, cells mount a complex signaling-based response that not only coordinates the repair of the broken DNA strand but also activates cell cycle checkpoints and, if necessary, induces cell death. The last decade has seen a flurry of studies that have identified RNA-binding proteins (RBPs) as novel regulators of the DSB response. While many of these RBPs have well-characterized roles in gene expression, it is becoming increasingly clear that they also have non-canonical functions in the DSB response that go well beyond transcription, splicing and mRNA processing. Here, we review the current understanding of how RBPs are integrated into the cellular response to DSBs and describe how these proteins directly participate in signal transduction, amplification and repair at damaged chromatin. In addition, we discuss the implications of an RBP-mediated DSB response for genome instability and age-associated diseases such as cancer and neurodegeneration.

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Topics: RNA-binding protein (61%), DNA repair (59%), Chromatin (56%) ... show more

4 Citations


Open accessJournal ArticleDOI: 10.1093/NAR/GKAB479
Wenjie Wang1, Kuan Li1, Zhuo Yang1, Quancan Hou1  +2 moreInstitutions (1)
Abstract: Proper repair of damaged DNA is crucial for genetic integrity and organismal survival. As semi-autonomous organelles, plastids have their own genomes whose integrity must be preserved. Several factors have been shown to participate in plastid DNA damage repair; however, the underlying mechanism remains unclear. Here, we elucidate a mechanism of homologous recombination (HR) repair in chloroplasts that involves R-loops. We find that the recombinase RecA1 forms filaments in chloroplasts during HR repair, but aggregates as puncta when RNA:DNA hybrids accumulate. ssDNA-binding proteins WHY1/3 and chloroplast RNase H1 AtRNH1C are recruited to the same genomic sites to promote HR repair. Depletion of AtRNH1C or WHY1/3 significantly suppresses the binding of RNA polymerase to the damaged DNA, thus reducing HR repair and modulating microhomology-mediated double-strand break repair. Furthermore, we show that DNA polymerase IB works with AtRNH1C genetically to complete the DNA damage repair process. This study reveals the positive role of R-loops in facilitating the activities of WHY1/3 and RecA1, which in turn secures HR repair and organellar development.

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Topics: DNA repair (65%), DNA damage (59%), DNA polymerase (58%) ... show more

3 Citations


Journal ArticleDOI: 10.1016/J.GDE.2021.06.009
Abstract: Helicases are in the spotlight of DNA metabolism and are critical for DNA repair in all domains of life. At their biochemical core, they bind and hydrolyze ATP, converting this energy to translocate unidirectionally, with different strand polarities and substrate binding specificities, along one strand of a nucleic acid. In doing so, DNA and RNA helicases separate duplex strands or remove nucleoprotein complexes, affecting DNA repair and the architecture of replication forks. In this review, we focus on recent advances on the roles and regulations of DNA helicases in homologous recombination repair, a critical pathway for mending damaged chromosomes and for ensuring genome integrity.

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Topics: Helicase (74%), DNA repair (63%), DNA (59%) ... show more

2 Citations


Open accessJournal ArticleDOI: 10.7554/ELIFE.69881
08 Jul 2021-eLife
Abstract: DNA double-strand breaks (DSBs) are the most harmful DNA lesions and their repair is crucial for cell viability and genome integrity. The readout of DSB repair may depend on whether DSBs occur at transcribed versus non-transcribed regions. Some studies have postulated that DNA-RNA hybrids form at DSBs to promote recombinational repair, but others have challenged this notion. To directly assess whether hybrids formed at DSBs promote or interfere with the recombinational repair, we have used plasmid and chromosomal-based systems for the analysis of DSB-induced recombination in Saccharomyces cerevisiae. We show that, as expected, DNA-RNA hybrid formation is stimulated at DSBs. In addition, mutations that promote DNA-RNA hybrid accumulation, such as hpr1∆ and rnh1∆ rnh201∆, cause high levels of plasmid loss when DNA breaks are induced at sites that are transcribed. Importantly, we show that high levels or unresolved DNA-RNA hybrids at the breaks interfere with their repair by homologous recombination. This interference is observed for both plasmid and chromosomal recombination and is independent of whether the DSB is generated by endonucleolytic cleavage or by DNA replication. These data support a model in which DNA-RNA hybrids form fortuitously at DNA breaks during transcription and need to be removed to allow recombinational repair, rather than playing a positive role.

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Topics: Homologous recombination (57%), DNA replication (53%), DNA (53%) ... show more

2 Citations


References
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55 results found


Open accessJournal ArticleDOI: 10.1038/NMETH.2019
01 Jul 2012-Nature Methods
Abstract: Fiji is a distribution of the popular open-source software ImageJ focused on biological-image analysis. Fiji uses modern software engineering practices to combine powerful software libraries with a broad range of scripting languages to enable rapid prototyping of image-processing algorithms. Fiji facilitates the transformation of new algorithms into ImageJ plugins that can be shared with end users through an integrated update system. We propose Fiji as a platform for productive collaboration between computer science and biology research communities.

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Topics: Software design (51%), Software (50%)

30,888 Citations


Open accessJournal ArticleDOI: 10.1038/NMETH.3047
01 Aug 2014-Nature Methods
Abstract: Genome-wide, targeted loss-of-function pooled screens using the CRISPR (clustered regularly interspaced short palindrome repeats)–associated nuclease Cas9 in human and mouse cells provide an alternative screening system to RNA interference (RNAi) and have been used to reveal new mechanisms in diverse biological models1-4. Previously, we used a Genome-scale CRISPR Knock-Out (GeCKO) library to identify loss-of-function mutations conferring vemurafenib resistance in a melanoma model1. However, initial lentiviral delivery systems for CRISPR screening had low viral titer or required a cell line already expressing Cas9, limiting the range of biological systems amenable to screening. Here, we sought to improve both the lentiviral packaging and choice of guide sequences in our original GeCKO library1, where a pooled library of synthesized oligonucleotides was cloned into a lentiviral backbone containing both the Streptococcus pyogenes Cas9 nuclease and the single guide RNA (sgRNA) scaffold. To create a new vector capable of producing higher-titer virus (lentiCRISPRv2), we made several modifications, including removal of one of the nuclear localization signals (NLS), human codon-optimization of the remaining NLS and P2A bicistronic linker sequences, and repositioning of the U6-driven sgRNA cassette (Fig. 1a). These changes resulted in a ~10-fold increase in functional viral titer over lentiCRISPRv11 (Fig. 1b). Figure 1 New lentiviral CRISPR designs produce viruses with higher functional titer. To further increase viral titer, we also cloned a two-vector system, in which Cas9 (lentiCas9-Blast) and sgRNA (lentiGuide-Puro) are delivered using separate viral vectors with distinct antibiotic selection markers (Fig. 1a). LentiGuide-Puro has a ~100-fold increase in functional viral titer over the original lentiCRISPRv1 (Fig. 1b). Both single and dual-vector systems mediate efficient knock-out of a genomically-integrated copy of EGFP in human cells (Supplementary Fig. 1). Whereas the dual vector system enables generation of Cas9-expressing cell lines which can be subsequently used for screens using lentiGuide-Puro, the single vector lentiCRISPRv2 may be better suited for in vivo or primary cell screening applications. In addition to the vector improvements, we designed and synthesized new human and mouse GeCKOv2 sgRNA libraries (Supplementary Methods) with several improvements (Table 1): First, for both human and mouse libraries, to target all genes with a uniform number of sgRNAs, we selected 6 sgRNAs per gene distributed over 3-4 constitutively expressed exons. Second, to further minimize off-target genome modification, we improved the calculation of off-target scores based on specificity analysis5. Third, to inactivate microRNAs (miRNAs) which play a key role in transcriptional regulation, we added sgRNAs to direct mutations to the pre-miRNA hairpin structure6. Finally, we targeted ~1000 additional genes not included in the original GeCKO library. Table 1 Comparison of new GeCKO v2 human and mouse sgRNA libraries with existing CRISPR libraries. Both libraries, mouse and human, are divided into 2 sub-libraries — containing 3 sgRNAs targeting each gene in the genome, as well as 1000 non-targeting control sgRNAs. Screens can be performed by combining both sub-libraries, yielding 6 sgRNAs per gene, for higher coverage. Alternatively, individual sub-libraries can be used in situations where cell numbers are limiting (eg. primary cells, in vivo screens). The human and mouse libraries have been cloned into lentiCRISPRv2 and into lentiGuide-Puro and deep sequenced to ensure uniform representation (Supplementary Fig. 2, 3). These new lentiCRISPR vectors and human and mouse libraries further improve the GeCKO reagents for diverse screening applications. Reagents are available to the academic community through Addgene and associated protocols, support forums, and computational tools are available via the Zhang lab website (www.genome-engineering.org).

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Topics: CRISPR interference (56%), Cas9 (55%), CRISPR (55%)

2,826 Citations


Book ChapterDOI: 10.1016/0076-6879(83)01015-0
Abstract: The one-step gene disruption techniques described here are versatile in that a disruption can be made simply by the appropriate cloning experiment. The resultant chromosomal insertion is nonreverting and contains a genetically linked marker. Detailed knowledge of the restriction map of a fragment is not necessary. It is even possible to "probe" a fragment that is unmapped for genetic functions by constructing a series of insertions and testing each one for its phenotype.

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Topics: Chromosomal Insertion (52%), Fungal genetics (50%), RAD52 Gene (50%)

2,825 Citations


Journal ArticleDOI: 10.1016/0092-8674(83)90331-8
01 May 1983-Cell
Abstract: Gene conversion is the nonreciprocal transfer of information from one DNA duplex to another; in meiosis, it is frequently associated with crossing-over. We review the genetic properties of meiotic recombination and previous models of conversion and crossing-over. In these models, recombination is initiated by single-strand nicks, and heteroduplex DNA is generated. Gene conversion is explained by the repair of mismatches present in heteroduplex DNA. We propose a new mechanism for meiotic recombination, in which events are initiated by double-strand breaks that are enlarged to double-strand gaps. Gene conversion can then occur by the repair of a double-strand gap, and postmeiotic segregation can result from heteroduplex DNA formed at the boundaries of the gap-repair region. The repair of double-strand gaps is an efficient process in yeast, and is known to be associated with crossing-over. The genetic implications of the double-strand-break repair model are explored.

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Topics: Meiotic gene conversion (68%), Meiotic recombination checkpoint (64%), RAD52 Gene (63%) ... show more

2,323 Citations


Open accessJournal ArticleDOI: 10.1016/S0092-8674(00)81876-0
07 Feb 1997-Cell
Abstract: Meiotic recombination in S. cerevisiae is initiated by double-strand breaks (DSBs). In certain mutants, breaks accumulate with a covalently attached protein, suggesting that cleavage is catalyzed by the DSB-associated protein via a topoisomerase-like transesterase mechanism. We have purified these protein-DNA complexes and identified the protein as Spo11, one of several proteins required for DSB formation. These findings strongly implicate Spo11 as the catalytic subunit of the meiotic DNA cleavage activity. This is the first identification of a biochemical function for any of the gene products involved in DSB formation. Spo11 defines a protein family with other members in fission yeast, nematodes, and archaebacteria. The S. pombe homolog, rec12p, is also known to be required for meiotic recombination. Thus, these findings provide direct evidence that the mechanism of meiotic recombination initiation is evolutionarily conserved.

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Topics: Meiotic DNA double-strand break formation (70%), Spo11 (69%), DMC1 (67%) ... show more

1,581 Citations


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