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iMUT-seq: high-resolution DSB-induced mutation profiling reveals prevalent homologous-recombination dependent mutagenesis
DNA double-strand breaks (DSBs) are highly mutagenic making them central to many pathologies. Here, the authors developed a highly sensitive sequencing approach to study DSB mutagenesis, yielding insights into mutagenic outcomes and characterising their underlying mechanisms.
- Aldo S. Bader
- & Martin Bushell
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Article
| Open Access
Genome-wide analysis of DNA-PK-bound MRN cleavage products supports a sequential model of DSB repair pathway choice
Deshpande et al show that MRN nuclease-dependent processing of DNA ends in human cells occurs at sites bound by DNA-PK. Chromatin immunoprecipitation analysis of DNA-PK, MRN, and CtIP supports a sequential model of pathway choice.
- Rajashree A. Deshpande
- , Alberto Marin-Gonzalez
- & Tanya T. Paull
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| Open Access
Ultra-deep sequencing validates safety of CRISPR/Cas9 genome editing in human hematopoietic stem and progenitor cells
As CRISPR-based therapies enter the clinic, evaluation of safety remains a critical and active area of study. Here the authors use next generation sequencing to achieve high sequencing depth and demonstrate that clinically relevant delivery of high-fidelity Cas9 to primary HSPCs and ex vivo culture up to 10 days does not introduce or enrich for tumorigenic variants.
- M. Kyle Cromer
- , Valentin V. Barsan
- & Matthew H. Porteus
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| Open Access
The importance of DNAPKcs for blunt DNA end joining is magnified when XLF is weakened
DNAPKcs and its kinase activity are required for blunt DNA break end joining when the bridging factor XLF is weakened, but for homologous recombination and radiation resistance, the influence of DNAPKcs is not further enhanced with loss of XLF.
- Metztli Cisneros-Aguirre
- , Felicia Wednesday Lopezcolorado
- & Jeremy M. Stark
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| Open Access
Coiled-coil heterodimer-based recruitment of an exonuclease to CRISPR/Cas for enhanced gene editing
The CRISPR/Cas system has emerged as a powerful and versatile genome engineering tool. Here the authors couple Cas9 to effector protein Exonuclease III via coiled-coil mediated interactions, termed CCExo, leading to increased deletion sizes and enhanced gene knock-out efficiencies in cell lines, primary cells and in vivo.
- Duško Lainšček
- , Vida Forstnerič
- & Roman Jerala
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| Open Access
Harnessing DSB repair to promote efficient homology-dependent and -independent prime editing
Prime editing is a next-generation approach for precision genome engineering. Here the authors design a nuclease-based prime editor that leverages DNA repair pathways for targeted genomic insertions.
- Martin Peterka
- , Nina Akrap
- & Marcello Maresca
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Article
| Open Access
Zinc finger protein ZNF384 is an adaptor of Ku to DNA during classical non-homologous end-joining
Classical non-homologous end-joining (cNHEJ) is the dominant pathway used by human cells to repair DNA double-strand breaks (DSBs) and maintain genome stability. Here the authors show that PARP1-driven chromatin expansion allows the recruitment of ZNF384, which in turn recruits Ku70/Ku80 to facilitate cNHEJ.
- Jenny Kaur Singh
- , Rebecca Smith
- & Haico van Attikum
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Article
| Open Access
Small tandem DNA duplications result from CST-guided Pol α-primase action at DNA break termini
Error-prone repair of DNA double-strand breaks have been implied to cause cancer-associated genome alterations, but the mechanism of their formation remains unclear. Here the authors find that DNA polymerase α primase plays part in tandem duplication formation at CRISPR/Cas9-induced complementary 3′ ssDNA protrusions.
- Joost Schimmel
- , Núria Muñoz-Subirana
- & Marcel Tijsterman
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Article
| Open Access
Mechanism of MRX inhibition by Rif2 at telomeres
Different proteins localised at telomeres ensure chromosome end stability to prevent double strand-end break recognition. Here the authors provide new insight into how in S. cerevisiae the interaction between Rif2 and Rad50 inhibits MRX functions at telomeres.
- Florian Roisné-Hamelin
- , Sabrina Pobiega
- & Stéphane Marcand
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| Open Access
CHD7 and 53BP1 regulate distinct pathways for the re-ligation of DNA double-strand breaks
Chromatin is dynamically remodeled in response to DNA damage in favour of repair. Here the authors reveal how the chromatin remodeler CHD7 and chromatin binding protein 53BP1 regulate distinct DNA repair pathways.
- Magdalena B. Rother
- , Stefania Pellegrino
- & Haico van Attikum
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Article
| Open Access
DNA double-strand breaks induce H2Ax phosphorylation domains in a contact-dependent manner
Formation of γH2Ax serves as a checkpoint for double-strand break (DSB) repair pathways. Here the authors reveal via integrated chromatin analysis that γH2Ax domains are established by chromosomal contacts with the DSB site.
- Patrick L. Collins
- , Caitlin Purman
- & Eugene M. Oltz
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Article
| Open Access
REV7 is required for processing AID initiated DNA lesions in activated B cells
REV7 has emerged as a critical regulator of DNA double-strand breaks repair. Here, the authors show that REV7 is crucial for both antibody class switch recombination and somatic hypermutation in activated B cells, in addition to their survival upon AID-deamination.
- Dingpeng Yang
- , Ying Sun
- & Fei-Long Meng
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| Open Access
Molecular basis for assembly of the shieldin complex and its implications for NHEJ
Shieldin, including SHLD1, SHLD2, SHLD3 and REV7, functions to suppress the DNA termini nucleolytic resection during non-homologous end joining (NHEJ). Here the authors present the crystal structure of the SHLD3-REV7-SHLD2 ternary complex revealing insights into the mechanism of the complex.
- Ling Liang
- , Jiawen Feng
- & Yuxin Yin
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| Open Access
FOXL2 directs DNA double-strand break repair pathways by differentially interacting with Ku
The Ku complex, formed by XRCC5/6 heterodimer, binds to double strand break (DSB) ends, initiating non homologous end joining (NHEJ) and preventing homologous recombination (HR). Here, the authors reveal that FOXL2, a forkhead family transcriptional factor, directs DSB repair pathway choice by acetylation-dependent binding to Ku.
- Hanyong Jin
- , Boeun Lee
- & Jeehyeon Bae
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| Open Access
The essential elements for the noncovalent association of two DNA ends during NHEJ synapsis
During a process termed synapsis, the two DNA ends at a double-strand break (DSB) are brought together into physical proximity. Here, the authors use a single-molecule FRET approach with purified proteins to investigate the mechanism of synapsis in DSB repair by non-homologous DNA end joining (NHEJ).
- Bailin Zhao
- , Go Watanabe
- & Michael R. Lieber
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| Open Access
Rif1 S-acylation mediates DNA double-strand break repair at the inner nuclear membrane
Rif1 is involved in different processes such as telomere homeostasis, DNA replication timing, and DNA double strand break (DSB) repair pathway choice. Here, the authors reveal that Rif1 S-acylation facilitates the accumulation of Rif1 at DSBs, attenuation of DNA end-resection, and DSB repair by non-homologous end-joining.
- Gabriele A. Fontana
- , Daniel Hess
- & Ulrich Rass
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Article
| Open Access
The ASCIZ-DYNLL1 axis promotes 53BP1-dependent non-homologous end joining and PARP inhibitor sensitivity
53BP1 is a key player in non-homologous end joining (NHEJ). Here the authors reveal an important role for the multifunctional homodimeric protein hub dynein light chain 1 (DYNLL1) in increasing the efficacy of 53BP1-mediated repair of DNA double-strand breaks (DSBs) by NHEJ.
- Jordan R. Becker
- , Raquel Cuella-Martin
- & J. Ross Chapman
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| Open Access
Pol μ dGTP mismatch insertion opposite T coupled with ligation reveals promutagenic DNA repair intermediate
Incorporation of mismatched nucleotides during DNA replication or repair can lead to mutagenesis. Here the authors reveal that DNA ligase can ligate NHEJ intermediates following incorporation of 8-oxodGTP or dGTP opposite T by DNA Polymerase mu (Pol mu) in vitro, which suggests that Pol mu could cause promutagenic mismatches during DSB repair.
- Melike Çağlayan
- & Samuel H. Wilson
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| Open Access
An OB-fold complex controls the repair pathways for DNA double-strand breaks
How repair pathway selection occurs is still a matter of debate and many factors have been associated to this function. Here the authors provide insight into the role of FAM35A and C20ORF196, two REV7-interacting proteins, which are recruited at double-strand breaks to promote non-homologous end joining repair.
- Shengxian Gao
- , Sumin Feng
- & Dongyi Xu
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Article
| Open Access
PAXX and its paralogs synergistically direct DNA polymerase λ activity in DNA repair
PAXX functions as part of the nonhomologous end-joining pathway to repair double-strand DNA breaks. Here the authors show PAXX and its paralogs interact with polymerase lambda to promote joining of incompatible ends.
- Andrew Craxton
- , Deeksha Munnur
- & Michal Malewicz
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| Open Access
Structures of DNA-bound human ligase IV catalytic core reveal insights into substrate binding and catalysis
DNA Ligase IV (LigIV) catalyzes nick sealing of DNA double-strand break substrates during non-homologous end-joining. Here the authors present the crystal structures of two human LigIV DNA-bound catalytic states, which provide insights into its catalytic mechanism and the molecular basis of LIG4 syndrome causing disease mutations.
- Andrea M. Kaminski
- , Percy P. Tumbale
- & Katarzyna Bebenek
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| Open Access
C-NHEJ without indels is robust and requires synergistic function of distinct XLF domains
Many factors are involved in end joining (EJ) repair of double strand breaks. Here the authors present a method to identify a chromosomal break repair event that requires classical non homologues end joining (C-NHEJ) using Cas9-based end joining tools, and define a role of CNHEJ factor XLF in repair.
- Ragini Bhargava
- , Manbir Sandhu
- & Jeremy M. Stark
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Article
| Open Access
MRN complex-dependent recruitment of ubiquitylated BLM helicase to DSBs negatively regulates DNA repair pathways
Bloom helicase is recruited to double strand breaks in an ATM dependent manner. Here the authors show that Bloom helicase is recruited to double strand breaks in an ATM and MRN dependent manner with HR and NHEJ regulated by the helicase depending on the phase of the cell cycle.
- Vivek Tripathi
- , Himanshi Agarwal
- & Sagar Sengupta
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Article
| Open Access
Inhibition of NHEJ repair by type II-A CRISPR-Cas systems in bacteria
The double-strand breaks generated by CRISPR-Cas systems are the target of multiple DNA repair pathways. Here the authors find incompatibility between NHEJ and type II-A CRISPR-Cas systems due to Csn2 mediated inhibition of end-joining.
- Aude Bernheim
- , Alicia Calvo-Villamañán
- & David Bikard
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Article
| Open Access
ATM and CDK2 control chromatin remodeler CSB to inhibit RIF1 in DSB repair pathway choice
Cockayne syndrome group B protein (CSB) is a multifunctional chromatin remodeler involved in double-strand break repair. Here the authors investigate the molecular post-translational signals regulating CSB activity.
- Nicole L. Batenburg
- , John R. Walker
- & Xu-Dong Zhu
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Article
| Open Access
Dual loss of human POLQ and LIG4 abolishes random integration
Homologous recombination mediated gene targeting is highly inefficient in human cells due to random integration events, Here the authors show that dual repression of polymerase θ and DNA ligase IV eliminate random integration events.
- Shinta Saito
- , Ryo Maeda
- & Noritaka Adachi
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| Open Access
Rad52 competes with Ku70/Ku86 for binding to S-region DSB ends to modulate antibody class-switch DNA recombination
Class switch DNA recombination (CSR) is critical for maturation of antibody response, and relies on Ku-mediated NHEJ of DSBs in the IgH S regions for recombination. This study shows Rad52 contributes to CSR through a Ku-independent alternative NHEJ that introduces microhomologies in S–S junctions.
- Hong Zan
- , Connie Tat
- & Paolo Casali
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Article
| Open Access
PAXX promotes KU accumulation at DNA breaks and is essential for end-joining in XLF-deficient mice
Non-homologous end-joining is the key pathway for repairing double-stranded DNA breaks in mammalian cells. Here the authors show that PAXX promotes the accumulation of KU at DNA breaks and is essential for end-joining in cells lacking XLF.
- Xiangyu Liu
- , Zhengping Shao
- & Shan Zha
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Article
| Open Access
CDK1 phosphorylates WRN at collapsed replication forks
End-resection of double strand DNA breaks is essential for pathway choice between non-homologous end-joining and homologous recombination. Here the authors show that phosphorylation of WRN helicase by CDK1 is essential for resection at replication-related breaks.
- Valentina Palermo
- , Sara Rinalducci
- & Pietro Pichierri
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Article
| Open Access
CRISPaint allows modular base-specific gene tagging using a ligase-4-dependent mechanism
The use of site-specific gene insertion is a powerful method for investigating gene function. Here the authors describe CRISPaint, a universal tagging system using CRISPR-Cas9 to insert genes in an NHEJ dependent manner.
- Jonathan L. Schmid-Burgk
- , Klara Höning
- & Veit Hornung
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Article
| Open Access
The Ku-binding motif is a conserved module for recruitment and stimulation of non-homologous end-joining proteins
Werner syndrome is a progeroid disease characterised by genetic instability due to mutations to the WRN helicase/exonuclease. Here the authors define a novel Ku binding motif (KBM) and show that two such motifs facilitate the involvement of WRN in DNA double-strand break repair.
- Gabrielle J. Grundy
- , Stuart L. Rulten
- & Keith W. Caldecott
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Article
| Open Access
RS-1 enhances CRISPR/Cas9- and TALEN-mediated knock-in efficiency
CRISPR/Cas9 and transcription activator-like effector nuclease (TALEN) are becoming major tools for genome editing. Here, Song et al. show that RS-1, a small-molecule enhancer for homology directed repair, increases the CRISPR/Cas9 and TALEN mediated knock-in efficiency both in vitro and in vivowith rabbit.
- Jun Song
- , Dongshan Yang
- & Jifeng Zhang
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| Open Access
Mutagenic consequences of a single G-quadruplex demonstrate mitotic inheritance of DNA replication fork barriers
Barriers to DNA replication are potent sources of genome instability. Here, the authors provide a mechanistic model for how a single persistent G-quadruplex structure generates multiple substrates for polymerase theta-mediated end-joining, thus causing multiple deletions during animal development.
- Bennie Lemmens
- , Robin van Schendel
- & Marcel Tijsterman
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Article
| Open Access
LRF maintains genome integrity by regulating the non-homologous end joining pathway of DNA repair
Leukemia/lymphoma-related factor (LRF), a transcriptional repressor, plays key roles in cell fate decision and tumorigenesis. Here, Liu et al. show that LRF loss results in defective classical non-homologous end joining, genomic instability and hypersensitivity to ionizing radiation, revealing a transcription-independent regulation of DNA-PK complex.
- Xue-Song Liu
- , Gurushankar Chandramouly
- & Pier Paolo Pandolfi
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Elucidation of IgH 3′ region regulatory role during class switch recombination via germline deletion
The molecular mechanisms of antibody class switching are incompletely understood. Here the authors show by using mice specifically lacking the IgH 3′ regulatory region enhancers that they prime the first steps of the class switch recombination.
- Alexis Saintamand
- , Pauline Rouaud
- & Yves Denizot
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Article
| Open Access
Interactome analysis identifies a new paralogue of XRCC4 in non-homologous end joining DNA repair pathway
DNA double-strand breaks (DSBs), a highly deleterious form of DNA damage, are associated with multiple types of broken ends. Here, the authors identify a XRCC4-like factor that functions in the non-homologous end-joining DNA repair pathway to repair DSBs with complex broken ends.
- Mengtan Xing
- , Mingrui Yang
- & Dongyi Xu
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TRIP13 promotes error-prone nonhomologous end joining and induces chemoresistance in head and neck cancer
Squamous cell carcinoma of the head and neck often becomes resistant to treatment. In this study, Banerjee et al. show that the ATPase TRIP13 can mediate resistance in cells from these tumours by inducing error prone non-homologous end joining DNA repair.
- Rajat Banerjee
- , Nickole Russo
- & Nisha J. D’Silva
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The fidelity of the ligation step determines how ends are resolved during nonhomologous end joining
DNA double strand breaks result in various types of damaged termini and are resolved by non-homologous end joining, but how cells coordinate the different steps that occur during repair is not clear. Here the authors show that a DNA ligase coordinates processing prior to the ligation step to limit errors.
- Crystal A. Waters
- , Natasha T. Strande
- & Dale A. Ramsden
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A histone H3K36 chromatin switch coordinates DNA double-strand break repair pathway choice
DNA double strand breaks are repaired by nonhomologous end-joining (NHEJ) or homologous recombination (HR) pathways. Here, Pai et al.discover that post-translational modification of lysine 36 of histone H3 plays a key role in determining double strand repair pathway choice.
- Chen-Chun Pai
- , Rachel S. Deegan
- & Timothy C. Humphrey
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DNA repair choice defines a common pathway for recruitment of chromatin regulators
Chromatin regulators facilitate repair of DNA double-strand breaks in chromosomal DNA. The authors show that the recruitment of such chromatin regulators to DNA lesions is controlled by the choice of DNA repair pathway.
- Gwendolyn Bennett
- , Manolis Papamichos-Chronakis
- & Craig L. Peterson
Structural role for DNA Ligase IV in promoting the fidelity of non-homologous end joining