RNF8 has both KU-dependent and independent roles in chromosomal break repair

Abstract Chromosomal double strand breaks (DSBs) can initiate several signaling events, such as ubiquitination, however the precise influence of such signaling on DSB repair outcomes remains poorly understood. With an RNA interference screen, we found that the E3 ubiquitin ligase RNF8 suppresses a deletion rearrangement mediated by canonical non-homologous end joining (C-NHEJ). We also found that RNF8 suppresses EJ without insertion/deletion mutations, which is a hallmark of C-NHEJ. Conversely, RNF8 promotes alternative EJ (ALT-EJ) events involving microhomology that is embedded from the edge of the DSB. These ALT-EJ events likely require limited end resection, whereas RNF8 is not required for single-strand annealing repair involving extensive end resection. Thus, RNF8 appears to specifically facilitate repair events requiring limited end resection, which we find is dependent on the DSB end protection factor KU. However, we also find that RNF8 is important for homology-directed repair (HDR) independently of KU, which appears linked to promoting PALB2 function. Finally, the influence of RNF8 on EJ is distinct from 53BP1 and the ALT-EJ factor, POLQ. We suggest that RNF8 mediates both ALT-EJ and HDR, but via distinct mechanisms, since only the former is dependent on KU.

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Necessities in the Processing of DNA Double Strand Breaks and Their Effects on Genomic Instability and Cancer

Cancers ◽

10.3390/cancers11111671 ◽

2019 ◽

Vol 11 (11) ◽

pp. 1671 ◽

Cited By ~ 15

Author(s):

George Iliakis ◽

Emil Mladenov ◽

Veronika Mladenova

Keyword(s):

High Speed ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

End Joining ◽

Strand Breaks ◽

Large Deletions ◽

Single Strand Annealing ◽

Modes Of Processing ◽

Non Homologous End Joining ◽

Strand Annealing

Double strand breaks (DSBs) are induced in the DNA following exposure of cells to ionizing radiation (IR) and are highly consequential for genome integrity, requiring highly specialized modes of processing. Erroneous processing of DSBs is a cause of cell death or its transformation to a cancer cell. Four mechanistically distinct pathways have evolved in cells of higher eukaryotes to process DSBs, providing thus multiple options for the damaged cells. The homologous recombination repair (HRR) dependent subway of gene conversion (GC) removes IR-induced DSBs from the genome in an error-free manner. Classical non-homologous end joining (c-NHEJ) removes DSBs with very high speed but is unable to restore the sequence at the generated junction and can catalyze the formation of translocations. Alternative end-joining (alt-EJ) operates on similar principles as c-NHEJ but is slower and more error-prone regarding both sequence preservation and translocation formation. Finally, single strand annealing (SSA) is associated with large deletions and may also form translocations. Thus, the four pathways available for the processing of DSBs are not alternative options producing equivalent outcomes. We discuss the rationale for the evolution of pathways with such divergent properties and fidelities and outline the logic and necessities that govern their engagement. We reason that cells are not free to choose one specific pathway for the processing of a DSB but rather that they engage a pathway by applying the logic of highest fidelity selection, adapted to necessities imposed by the character of the DSB being processed. We introduce DSB clusters as a particularly consequential form of chromatin breakage and review findings suggesting that this form of damage underpins the increased efficacy of high linear energy transfer (LET) radiation modalities. The concepts developed have implications for the protection of humans from radon-induced cancer, as well as the treatment of cancer with radiations of high LET.

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Rejoining of DNA Double-Strand Breaks as a Function of Overhang Length

Molecular and Cellular Biology ◽

10.1128/mcb.25.3.896-906.2005 ◽

2005 ◽

Vol 25 (3) ◽

pp. 896-906 ◽

Cited By ~ 71

Author(s):

James M. Daley ◽

Thomas E. Wilson

Keyword(s):

Gc Content ◽

Double Strand Breaks ◽

Nonhomologous End Joining ◽

Dna Double Strand Breaks ◽

End Joining ◽

Strand Breaks ◽

Single Strand Annealing ◽

Primary Determinant ◽

Overhang Length ◽

Strand Annealing

ABSTRACT The ends of spontaneously occurring double-strand breaks (DSBs) may contain various lengths of single-stranded DNA, blocking lesions, and gaps and flaps generated by end annealing. To investigate the processing of such structures, we developed an assay in which annealed oligonucleotides are ligated onto the ends of a linearized plasmid which is then transformed into Saccharomyces cerevisiae. Reconstitution of a marker occurs only when the oligonucleotides are incorporated and repair is in frame, permitting rapid analysis of complex DSB ends. Here, we created DSBs with compatible overhangs of various lengths and asked which pathways are required for their precise repair. Three mechanisms of rejoining were observed, regardless of overhang polarity: nonhomologous end joining (NHEJ), a Rad52-dependent single-strand annealing-like pathway, and a third mechanism independent of the first two mechanisms. DSBs with overhangs of less than 4 bases were mainly repaired by NHEJ. Repair became less dependent on NHEJ when the overhangs were longer or had a higher GC content. Repair of overhangs greater than 8 nucleotides was as much as 150-fold more efficient, impaired 10-fold by rad52 mutation, and highly accurate. Reducing the microhomology extent between long overhangs reduced their repair dramatically, to less than NHEJ of comparable short overhangs. These data support a model in which annealing energy is a primary determinant of the rejoining efficiency and mechanism.

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The Role of Nonhomologous DNA End Joining, Conservative Homologous Recombination, and Single-Strand Annealing in the Cell Cycle-Dependent Repair of DNA Double-Strand Breaks Induced by H2O2in Mammalian Cells

Radiation Research ◽

10.1667/rr1375.1 ◽

2008 ◽

Vol 170 (6) ◽

pp. 784-793 ◽

Cited By ~ 14

Author(s):

Marlis Frankenberg-Schwager ◽

Manuela Becker ◽

Irmgard Garg ◽

Elke Pralle ◽

Hartmut Wolf ◽

...

Keyword(s):

Cell Cycle ◽

Mammalian Cells ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

End Joining ◽

Strand Breaks ◽

Single Strand Annealing ◽

Strand Annealing ◽

Cycle Dependent

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Simultaneous precise editing of multiple genes in human cells

Nucleic Acids Research ◽

10.1093/nar/gkz669 ◽

2019 ◽

Vol 47 (19) ◽

pp. e116-e116 ◽

Cited By ~ 17

Author(s):

Stephan Riesenberg ◽

Manjusha Chintalapati ◽

Dominik Macak ◽

Philipp Kanis ◽

Tomislav Maricic ◽

...

Keyword(s):

Genome Editing ◽

Kinase Inhibitor ◽

Double Strand Breaks ◽

End Joining ◽

Nucleotide Substitutions ◽

Strand Breaks ◽

Homology Directed Repair ◽

A Genome ◽

Dependent Protein ◽

Non Homologous End Joining

Abstract When double-strand breaks are introduced in a genome by CRISPR they are repaired either by non-homologous end joining (NHEJ), which often results in insertions or deletions (indels), or by homology-directed repair (HDR), which allows precise nucleotide substitutions to be introduced if a donor oligonucleotide is provided. Because NHEJ is more efficient than HDR, the frequency with which precise genome editing can be achieved is so low that simultaneous editing of more than one gene has hitherto not been possible. Here, we introduced a mutation in the human PRKDC gene that eliminates the kinase activity of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). This results in an increase in HDR irrespective of cell type and CRISPR enzyme used, sometimes allowing 87% of chromosomes in a population of cells to be precisely edited. It also allows for precise editing of up to four genes simultaneously (8 chromosomes) in the same cell. Transient inhibition of DNA-PKcs by the kinase inhibitor M3814 is similarly able to enhance precise genome editing.

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Recent Perspectives in Radiation-Mediated DNA Damage and Repair: Role of NHEJ and Alternative Pathways

10.5772/intechopen.96374 ◽

2021 ◽

Author(s):

Ajay Kumar Sharma ◽

Priyanka Shaw ◽

Aman Kalonia ◽

M.H. Yashavarddhan ◽

Pankaj Chaudhary ◽

...

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Double Strand Breaks ◽

Single Strand ◽

End Joining ◽

Strand Breaks ◽

Single Strand Annealing ◽

Repair Pathways ◽

Non Homologous End Joining ◽

Alternative Nhej

Radiation is one of the causative agents for the induction of DNA damage in biological systems. There is various possibility of radiation exposure that might be natural, man-made, intentional, or non-intentional. Published literature indicates that radiation mediated cell death is primarily due to DNA damage that could be a single-strand break, double-strand breaks, base modification, DNA protein cross-links. The double-strand breaks are lethal damage due to the breakage of both strands of DNA. Mammalian cells are equipped with strong DNA repair pathways that cover all types of DNA damage. One of the predominant pathways that operate DNA repair is a non-homologous end-joining pathway (NHEJ) that has various integrated molecules that sense, detect, mediate, and repair the double-strand breaks. Even after a well-coordinated mechanism, there is a strong possibility of mutation due to the flexible nature in joining the DNA strands. There are alternatives to NHEJ pathways that can repair DNA damage. These pathways are alternative NHEJ pathways and single-strand annealing pathways that also displayed a role in DNA repair. These pathways are not studied extensively, and many reports are showing the relevance of these pathways in human diseases. The chapter will very briefly cover the radiation, DNA repair, and Alternative repair pathways in the mammalian system. The chapter will help the readers to understand the basic and applied knowledge of radiation mediated DNA damage and its repair in the context of extensively studied NHEJ pathways and unexplored alternative NHEJ pathways.

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The non-homologous end joining factor Ku orchestrates replication fork resection and fine-tunes Rad51-mediated fork restart

10.1101/162677 ◽

2017 ◽

Author(s):

Ana Teixeira-Silva ◽

Anissia Ait Saada ◽

Ismail Iraqui ◽

Marina Charlotte Nocente ◽

Karine Fréon ◽

...

Keyword(s):

Replication Fork ◽

Double Strand Breaks ◽

Fine Tuning ◽

List Type ◽

End Joining ◽

Strand Breaks ◽

Replication Restart ◽

Dna End Resection ◽

Non Homologous End Joining ◽

End Resection

AbstractReplication requires Homologous Recombination (HR) to stabilize and restart terminally-arrested forks. HR-mediated fork processing requires single stranded DNA (ssDNA) gaps and not necessarily Double Strand Breaks. We used genetic and molecular assays to investigate fork-resection and restart at dysfunctional, unbroken forks in Schizosaccharomyces pombe. We found that fork-resection is a two-step process coordinated by the non-homologous end joining factor Ku. An initial resection mediated by MRN/Ctp1 removes Ku from terminally-arrested forks, generating ~ 110 bp sized gaps obligatory for subsequent Exo1-mediated long-range resection and replication restart. The lack of Ku results in slower fork restart, excessive resection, and impaired RPA recruitment. We propose that terminally-arrested forks undergo fork reversal, providing a single DNA end for Ku binding which primes RPA-coated ssDNA. We uncover an unprecedented role for Ku in orchestrating resection of unbroken forks and in fine-tuning HR-mediated replication restart.Ku orchestrates a two-steps DNA end-resection of terminally-arrested and unbroken forksMRN/Ctp1 removes Ku from terminally-arrested forks to initiate fork-resectiona ~110 bp sized ssDNA gap is sufficient and necessary to promote fork restart.The lack of Ku decreases ssDNA RPA-coating, and slows down replication fork restart.

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Competing roles of DNA end resection and non-homologous end joining functions in the repair of replication-born double-strand breaks by sister-chromatid recombination

Nucleic Acids Research ◽

10.1093/nar/gks1274 ◽

2012 ◽

Vol 41 (3) ◽

pp. 1669-1683 ◽

Cited By ~ 12

Author(s):

Sandra Muñoz-Galván ◽

Ana López-Saavedra ◽

Stephen P. Jackson ◽

Pablo Huertas ◽

Felipe Cortés-Ledesma ◽

...

Keyword(s):

Sister Chromatid ◽

Double Strand Breaks ◽

End Joining ◽

Strand Breaks ◽

Dna End Resection ◽

Non Homologous End Joining ◽

End Resection ◽

Sister Chromatid Recombination

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Antagonistic relationship of NuA4 with the Non-Homologous End-Joining machinery at DNA damage sites

10.1101/2021.01.29.428810 ◽

2021 ◽

Author(s):

Salar Ahmad ◽

Valerie Côté ◽

Xue Cheng ◽

Gaëlle Bourriquen ◽

Vasileia Sapountzi ◽

...

Keyword(s):

Budding Yeast ◽

Double Strand Breaks ◽

Repair Pathway ◽

Dna Double Strand Breaks ◽

End Joining ◽

Strand Breaks ◽

Dna End Resection ◽

Non Homologous End Joining ◽

Relationship Of ◽

End Resection

AbstractThe NuA4 histone acetyltransferase complex, apart from its known role in gene regulation, has also been directly implicated in the repair of DNA double-strand breaks (DSBs), favoring homologous recombination (HR) in S/G2 during the cell cycle. Here, we investigate the antagonistic relationship of NuA4 with non-homologous end joining (NHEJ) factors. We show that budding yeast Rad9, the 53BP1 ortholog, can inhibit NuA4 acetyltransferase activity when bound to chromatin in vitro. While we previously reported that NuA4 is recruited at DSBs during the S/G2 phase, we can also detect its recruitment in G1 when genes for NHEJ factors Rad9, Yku80 and Nej1 are mutated. This is accompanied with the binding of single-strand DNA binding protein RPA and Rad52, indicating DNA end resection in G1 as well as recruitment of the HR machinery. This NuA4 recruitment to DSBs in G1 depends on both Xrs2 and Lcd1/Ddc2. Introducing an acetyltransferase defective allele in these NHEJ mutant backgrounds decreases their hyper-resection phenotype in G1. Interestingly, we identified two novel non-histone acetylation targets of NuA4, Nej1 and Yku80. Acetyl-mimicking mutant of Nej1 inhibits repair of DNA breaks by NHEJ, decreases its interaction with other core NHEJ factors such as Yku80 and Lif1 and favors end resection. Altogether, these results establish a strong reciprocal antagonistic regulatory function of NuA4 and NHEJ factors in repair pathway choice and suggests a role of NuA4 in alternative repair mechanism that involves DNA-end resection in G1.Author SummaryDNA double-strand breaks (DSBs) are one of the most harmful form of DNA damage. Cells employ two major repair pathways to resolve DSBs: Homologous Recombination (HR) and Non-Homologous End Joining (NHEJ). Here we wanted to dissect further the role played by the NuA4 (Nucleosome acetyltransferase of histone H4) complex in the repair of DSBs. Budding yeast NuA4 complex, like its mammalian homolog TIP60 complex, has been shown to favor repair by HR. Here, we show that indeed budding yeast NuA4 and components of the NHEJ repair pathway share an antagonistic relationship. Deletion of NHEJ components enables increased recruitment of NuA4 in the vicinity of DSBs, where NuA4 favors the end resection process which is an underlying mechanism for HR repair. We also describe two independent modes responsible for the recruitment of NuA4 to DSB sites. Additionally, we also present two NHEJ core components as new targets of NuA4 acetyltransferase activity and suggest that these acetylation events can disassemble the NHEJ repair complex from DSBs, favoring repair by HR. Our study demonstrates the importance of NuA4 in the modulation of DSB repair pathway choice.

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