The RNF8 and RNF168 Ubiquitin Ligases Regulate Pro- and Anti-Resection Activities at Broken DNA Ends During Non-Homologous End Joining

Abstract As its name implies, the DNA dependent protein kinase (DNA-PK) requires DNA double-stranded ends for enzymatic activation. Here, I demonstrate that hairpinned DNA ends are ineffective for activating the kinase toward many of its well-studied substrates (p53, XRCC4, XLF, HSP90). However, hairpinned DNA ends robustly stimulate certain DNA-PK autophosphorylations. Specifically, autophosphorylation sites within the ABCDE cluster are robustly phosphorylated when DNA-PK is activated by hairpinned DNA ends. Of note, phosphorylation of the ABCDE sites is requisite for activation of the Artemis nuclease that associates with DNA-PK to mediate hairpin opening. This finding suggests a multi-step mechanism of kinase activation. Finally, I find that all non-homologous end joining (NHEJ) defective cells (whether deficient in components of the DNA-PK complex or components of the ligase complex) are similarly deficient in joining DNA double-stranded breaks (DSBs) with hairpinned termini.

Download Full-text

Characterization of gamma irradiation-induced mutations in Arabidopsis mutants deficient in non-homologous end joining

Journal of Radiation Research ◽

10.1093/jrr/rraa059 ◽

2020 ◽

Vol 61 (5) ◽

pp. 639-647

Author(s):

Yan Du ◽

Yoshihiro Hase ◽

Katsuya Satoh ◽

Naoya Shikazono

Keyword(s):

Gamma Rays ◽

Wild Type ◽

End Joining ◽

Complex Type ◽

Single Base ◽

In The Wild ◽

Non Homologous End Joining ◽

Dna Ends ◽

Wild Type Control

Abstract To investigate the involvement of the non-homologous end joining (NHEJ) pathway in plant mutagenesis by ionizing radiation, we conducted a genome-wide characterization of the mutations induced by gamma rays in NHEJ-deficient Arabidopsis mutants (AtKu70−/− and AtLig4−/−). Although both mutants were more sensitive to gamma rays than the wild-type control, the AtKu70−/− mutant was slightly more sensitive than the AtLig4−/− mutant. Single-base substitutions (SBSs) were the predominant mutations in the wild-type control, whereas deletions (≥2 bp) and complex-type mutations [i.e. more than two SBSs or short insertion and deletions (InDels) separated by fewer than 10 bp] were frequently induced in the mutants. Single-base deletions were the most frequent deletions in the wild-type control, whereas the most common deletions in the mutants were 11–30 bp. The apparent microhomology at the rejoined sites of deletions peaked at 2 bp in the wild-type control, but was 3–4 bp in the mutants. This suggests the involvement of alternative end joining and single-strand annealing pathways involving increased microhomology for rejoining DNA ends. Complex-type mutations comprising short InDels were frequently detected in the mutants, but not in the wild-type control. Accordingly, NHEJ is more precise than the backup pathways, and is the main pathway for rejoining the broken DNA ends induced by ionizing radiation in plants.

Download Full-text

Non-homologous End Joining Requires That the DNA-PK Complex Undergo an Autophosphorylation-dependent Rearrangement at DNA Ends

Journal of Biological Chemistry ◽

10.1074/jbc.m406432200 ◽

2004 ◽

Vol 279 (38) ◽

pp. 39408-39413 ◽

Cited By ~ 88

Author(s):

Yeturu V. R. Reddy ◽

Qi Ding ◽

Susan P. Lees-Miller ◽

Katheryn Meek ◽

Dale A. Ramsden

Keyword(s):

End Joining ◽

Non Homologous End Joining ◽

Dna Ends

Download Full-text

Structural mechanism of DNA-end synapsis in the non-homologous end joining pathway for repairing double-strand breaks: bridge over troubled ends

Biochemical Society Transactions ◽

10.1042/bst20180518 ◽

2019 ◽

Vol 47 (6) ◽

pp. 1609-1619 ◽

Cited By ~ 5

Author(s):

Qian Wu

Keyword(s):

Single Molecule ◽

Genome Instability ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

End Joining ◽

Structural Mechanism ◽

Strand Breaks ◽

Single Molecule Studies ◽

Non Homologous End Joining ◽

Dna Ends

Non-homologous end joining (NHEJ) is a major repair pathway for DNA double-strand breaks (DSBs), which is the most toxic DNA damage in cells. Unrepaired DSBs can cause genome instability, tumorigenesis or cell death. DNA end synapsis is the first and probably the most important step of the NHEJ pathway, aiming to bring two broken DNA ends close together and provide structural stability for end processing and ligation. This process is mediated through a group of NHEJ proteins forming higher-order complexes, to recognise and bridge two DNA ends. Spatial and temporal understanding of the structural mechanism of DNA-end synapsis has been largely advanced through recent structural and single-molecule studies of NHEJ proteins. This review focuses on core NHEJ proteins that mediate DNA end synapsis through their unique structures and interaction properties, as well as how they play roles as anchor and linker proteins during the process of ‘bridge over troubled ends'.

Download Full-text

XLF acts as a flexible connector during non-homologous end joining

10.1101/2020.07.29.227033 ◽

2020 ◽

Author(s):

Sean M. Carney ◽

Andrew T. Moreno ◽

Sadie C. Piatt ◽

Metztli Cisneros-Aguirre ◽

Felicia Wednesday Lopezcolorado ◽

...

Keyword(s):

Single Molecule ◽

Critical Factor ◽

Tail Length ◽

Binding Motif ◽

Dna Double Strand Breaks ◽

End Joining ◽

Synaptic Complex ◽

Intrinsically Disordered ◽

Non Homologous End Joining ◽

Dna Ends

AbstractNon-homologous end joining (NHEJ) is the predominant pathway that repairs DNA double strand breaks in vertebrates. During NHEJ DNA ends are held together by a multi-protein synaptic complex until they are ligated. Here we investigate the role of the intrinsically disordered C-terminal tail of XLF, a critical factor in end synapsis. We demonstrate that the XLF tail along with the Ku binding motif (KBM) at the extreme C-terminus are required for end joining. While the underlying sequence of the tail can be varied, a minimal tail length is required for NHEJ. Single-molecule FRET experiments that observe end synapsis in real-time show that this defect is due to a failure to closely align DNA ends. Our data supports a model in which a single C-terminal tail tethers XLF to Ku while allowing XLF to form interactions with XRCC4 that enable synaptic complex formation.

Download Full-text

Two-Stage Synapsis of DNA Ends during Non-homologous End Joining

Molecular Cell ◽

10.1016/j.molcel.2016.02.010 ◽

2016 ◽

Vol 61 (6) ◽

pp. 850-858 ◽

Cited By ~ 67

Author(s):

Thomas G.W. Graham ◽

Johannes C. Walter ◽

Joseph J. Loparo

Keyword(s):

End Joining ◽

Two Stage ◽

Non Homologous End Joining ◽

Dna Ends

Download Full-text

To Join or Not to Join: Decision Points Along the Pathway to Double-Strand Break Repair vs. Chromosome End Protection

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.708763 ◽

2021 ◽

Vol 9 ◽

Author(s):

Stephanie M. Ackerson ◽

Carlan Romney ◽

P. Logan Schuck ◽

Jason A. Stewart

Keyword(s):

Genome Stability ◽

Strand Break ◽

Dna Double Strand Breaks ◽

End Joining ◽

Dsb Repair ◽

Strand Breaks ◽

Decision Points ◽

Chromosome Fusions ◽

Non Homologous End Joining ◽

Dna Ends

The regulation of DNA double-strand breaks (DSBs) and telomeres are diametrically opposed in the cell. DSBs are considered one of the most deleterious forms of DNA damage and must be quickly recognized and repaired. Telomeres, on the other hand, are specialized, stable DNA ends that must be protected from recognition as DSBs to inhibit unwanted chromosome fusions. Decisions to join DNA ends, or not, are therefore critical to genome stability. Yet, the processing of telomeres and DSBs share many commonalities. Accordingly, key decision points are used to shift DNA ends toward DSB repair vs. end protection. Additionally, DSBs can be repaired by two major pathways, namely homologous recombination (HR) and non-homologous end joining (NHEJ). The choice of which repair pathway is employed is also dictated by a series of decision points that shift the break toward HR or NHEJ. In this review, we will focus on these decision points and the mechanisms that dictate end protection vs. DSB repair and DSB repair choice.

Download Full-text

XLF acts as a flexible connector during non-homologous end joining

eLife ◽

10.7554/elife.61920 ◽

2020 ◽

Vol 9 ◽

Cited By ~ 1

Author(s):

Sean M Carney ◽

Andrew T Moreno ◽

Sadie C Piatt ◽

Metztli Cisneros-Aguirre ◽

Felicia Wednesday Lopezcolorado ◽

...

Keyword(s):

Single Molecule ◽

Critical Factor ◽

Tail Length ◽

Binding Motif ◽

Dna Double Strand Breaks ◽

End Joining ◽

Synaptic Complex ◽

Intrinsically Disordered ◽

Non Homologous End Joining ◽

Dna Ends

Non-homologous end joining (NHEJ) is the predominant pathway that repairs DNA double-strand breaks in vertebrates. During NHEJ DNA ends are held together by a multi-protein synaptic complex until they are ligated. Here, we use Xenopus laevis egg extract to investigate the role of the intrinsically disordered C-terminal tail of the XRCC4-like factor (XLF), a critical factor in end synapsis. We demonstrate that the XLF tail along with the Ku-binding motif (KBM) at the extreme C-terminus are required for end joining. Although the underlying sequence of the tail can be varied, a minimal tail length is required for NHEJ. Single-molecule FRET experiments that observe end synapsis in real-time show that this defect is due to a failure to closely align DNA ends. Our data supports a model in which a single C-terminal tail tethers XLF to Ku, while allowing XLF to form interactions with XRCC4 that enable synaptic complex formation.

Download Full-text

ATM antagonizes NHEJ proteins assembly and DNA-ends synapsis at single-ended DNA double strand breaks

Nucleic Acids Research ◽

10.1093/nar/gkaa723 ◽

2020 ◽

Vol 48 (17) ◽

pp. 9710-9723

Author(s):

Sébastien Britton ◽

Pauline Chanut ◽

Christine Delteil ◽

Nadia Barboule ◽

Philippe Frit ◽

...

Keyword(s):

Dna Repair ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

End Joining ◽

Strand Breaks ◽

Repair Pathways ◽

Non Homologous End Joining ◽

Dna Strand ◽

Dna Ends

Abstract Two DNA repair pathways operate at DNA double strand breaks (DSBs): non-homologous end-joining (NHEJ), that requires two adjacent DNA ends for ligation, and homologous recombination (HR), that resects one DNA strand for invasion of a homologous duplex. Faithful repair of replicative single-ended DSBs (seDSBs) is mediated by HR, due to the lack of a second DNA end for end-joining. ATM stimulates resection at such breaks through multiple mechanisms including CtIP phosphorylation, which also promotes removal of the DNA-ends sensor and NHEJ protein Ku. Here, using a new method for imaging the recruitment of the Ku partner DNA-PKcs at DSBs, we uncover an unanticipated role of ATM in removing DNA-PKcs from seDSBs in human cells. Phosphorylation of DNA-PKcs on the ABCDE cluster is necessary not only for DNA-PKcs clearance but also for the subsequent MRE11/CtIP-dependent release of Ku from these breaks. We propose that at seDSBs, ATM activity is necessary for the release of both Ku and DNA-PKcs components of the NHEJ apparatus, and thereby prevents subsequent aberrant interactions between seDSBs accompanied by DNA-PKcs autophosphorylation and detrimental commitment to Lig4-dependent end-joining.

Download Full-text

C-Terminal Extensions of Ku70 and Ku80 Differentially Influence DNA End Binding Properties

International Journal of Molecular Sciences ◽

10.3390/ijms21186725 ◽

2020 ◽

Vol 21 (18) ◽

pp. 6725

Author(s):

Takabumi Inagawa ◽

Thomas Wennink ◽

Joyce H. G. Lebbink ◽

Guido Keijzers ◽

Bogdan I. Florea ◽

...

Keyword(s):

Bound States ◽

Protein Complexes ◽

Release Kinetics ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Strand Break ◽

End Joining ◽

Non Homologous End Joining ◽

Stable Association ◽

Dna Ends

The Ku70/80 heterodimer binds to DNA ends and attracts other proteins involved in the non-homologous end-joining (NHEJ) pathway of DNA double-strand break repair. We developed a novel assay to measure DNA binding and release kinetics using differences in Förster resonance energy transfer (FRET) of the ECFP-Ku70/EYFP-Ku80 heterodimer in soluble and DNA end bound states. We confirmed that the relative binding efficiencies of various DNA substrates (blunt, 3 nucleotide 5′ extension, and DNA hairpin) measured in the FRET assay reflected affinities obtained from direct measurements using surface plasmon resonance. The FRET assay was subsequently used to investigate Ku70/80 behavior in the context of a DNA-dependent kinase (DNA-PK) holocomplex. As expected, this complex was much more stable than Ku70/80 alone, and its stability was influenced by DNA-PK phosphorylation status. Interestingly, the Ku80 C-terminal extension contributed to DNA-PK complex stability but was not absolutely required for its formation. The Ku70 C-terminal SAP domain, on the other hand, was required for the stable association of Ku70/80 to DNA ends, but this effect was abrogated in DNA-PK holocomplexes. We conclude that FRET measurements can be used to determine Ku70/80 binding kinetics. The ability to do this in complex mixtures makes this assay particularly useful to study larger NHEJ protein complexes on DNA ends.

Download Full-text