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

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.

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

eLife ◽  
2020 ◽  
Vol 9 ◽  
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.

scholarly journals Non-homologous end joining minimizes errors by coordinating DNA processing with ligation

2019 ◽  
Author(s):  
Benjamin M. Stinson ◽  
Andrew T. Moreno ◽  
Johannes C. Walter ◽  
Joseph J. Loparo
Keyword(s):  
Single Molecule ◽  
Genome Stability ◽  
Short Range ◽  
Genome Instability ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Synaptic Complex ◽  
Processing Enzymes ◽  
Non Homologous End Joining ◽  
Dna Ends

Genome stability requires efficient and faithful repair of DNA double-strand breaks (DSBs). The predominant DSB repair pathway in human cells is non-homologous end-joining (NHEJ), which directly ligates DNA ends1–5. Broken DNA ends at DSBs are chemically diverse, and many are not compatible for direct ligation by the NHEJ-associated DNA Ligase IV (Lig4). To solve this problem, NHEJ end-processing enzymes including polymerases and nucleases modify ends until they are ligatable. How cells regulate end processing to minimize unnecessary genomic alterations6 during repair of pathological DSBs remains unknown. Using a biochemical system that recapitulates key features of cellular NHEJ, we previously demonstrated that DNA ends are initially tethered at a distance, followed by Lig4-mediated formation of a “short-range synaptic complex” in which DNA ends are closely aligned for ligation7. Here, we show that a wide variety of end-processing activities all depend on formation of the short-range complex. Moreover, using real-time single molecule imaging, we find that end processing occurs within the short-range complex. Confining end processing to the Lig4-dependent short-range synaptic complex promotes immediate ligation of compatible ends and ensures that incompatible ends are ligated as soon as they become compatible, thereby minimizing end processing. Our results elucidate how NHEJ exploits end processing to achieve versatility while minimizing errors that cause genome instability.

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

2019 ◽  
Vol 47 (6) ◽  
pp. 1609-1619 ◽  
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'.

scholarly journals SIRT6 is a DNA double-strand break sensor

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Lior Onn ◽  
Miguel Portillo ◽  
Stefan Ilic ◽  
Gal Cleitman ◽  
Daniel Stein ◽  
...  
Keyword(s):  
Dna Damage ◽  
Binding Capacity ◽  
Critical Factor ◽  
Strand Break ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Downstream Signaling ◽  
The Road ◽  
Dna Double Strand Break ◽  
Non Homologous End Joining

DNA double-strand breaks (DSB) are the most deleterious type of DNA damage. In this work, we show that SIRT6 directly recognizes DNA damage through a tunnel-like structure that has high affinity for DSB. SIRT6 relocates to sites of damage independently of signaling and known sensors. It activates downstream signaling for DSB repair by triggering ATM recruitment, H2AX phosphorylation and the recruitment of proteins of the homologous recombination and non-homologous end joining pathways. Our findings indicate that SIRT6 plays a previously uncharacterized role as a DNA damage sensor, a critical factor in initiating the DNA damage response (DDR). Moreover, other Sirtuins share some DSB-binding capacity and DDR activation. SIRT6 activates the DDR before the repair pathway is chosen, and prevents genomic instability. Our findings place SIRT6 as a sensor of DSB, and pave the road to dissecting the contributions of distinct DSB sensors in downstream signaling.

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

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.

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

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.

scholarly journals The DNAPKcs long-range C-NHEJ complex is required for blunt DNA end joining when XLF is weakened

2021 ◽  
Author(s):  
Metztli Cisneros-Aguirre ◽  
Felicia Wednesday Lopezcolorado ◽  
Linda Jillianne Tsai ◽  
Ragini Bhargava ◽  
Jeremy M Stark
Keyword(s):  
Long Range ◽  
De Novo ◽  
Dna Double Strand Breaks ◽  
Genome Maintenance ◽  
End Joining ◽  
Strand Breaks ◽  
Homology Directed Repair ◽  
Non Homologous End Joining ◽  
Dna Ends

Canonical non-homologous end joining (C-NHEJ) factors can assemble into a long-range (LR) complex with DNA ends relatively far apart that contains DNAPKcs, XLF, XRCC4, LIG4, and the KU heterodimer and a short-range (SR) complex lacking DNAPKcs that has the ends positioned for ligation. Since the SR complex can form de novo, the role of the LR complex (i.e., DNAPKcs) for chromosomal EJ is unclear. We have examined EJ of chromosomal blunt DNA double-strand breaks (DSBs), and found that DNAPKcs is significantly less important than XLF and XRCC4 for such EJ. However, weakening XLF via disrupting interaction interfaces (e.g., disrupting the XLF homodimer interface) causes a marked requirement for DNAPKcs, its kinase activity, and its ABCDE-cluster autophosphorylation sites for blunt DSB EJ. In contrast, other aspects of genome maintenance are sensitive to DNAPKcs kinase inhibition in a manner that is not further enhanced by XLF loss (i.e., suppression of homology-directed repair and structural variants, and IR-resistance). We suggest that DNAPKcs is required to position a weakened XLF in an LR complex that can transition into a functional SR complex for blunt DSB EJ, but also has distinct functions for other aspects of genome maintenance.

scholarly journals Dynamics of Ku and bacterial non-homologous end-joining characterized using single DNA molecule analysis

2021 ◽  
Author(s):  
Robin Öz ◽  
Jing L Wang ◽  
Raphael Guerois ◽  
Gaurav Goyal ◽  
Sriram KK ◽  
...  
Keyword(s):  
Single Molecule ◽  
End Joining ◽  
Complementary Dna ◽  
Dna Molecule ◽  
Dna Junctions ◽  
Non Homologous End Joining ◽  
Single Dna Molecule ◽  
Dna Ends ◽  
Shed Light

Abstract We use single-molecule techniques to characterize the dynamics of prokaryotic DNA repair by non-homologous end-joining (NHEJ), a system comprised only of the dimeric Ku and Ligase D (LigD). The Ku homodimer alone forms a ∼2 s synapsis between blunt DNA ends that is increased to ∼18 s upon addition of LigD, in a manner dependent on the C-terminal arms of Ku. The synapsis lifetime increases drastically for 4 nt complementary DNA overhangs, independently of the C-terminal arms of Ku. These observations are in contrast to human Ku, which is unable to bridge either of the two DNA substrates. We also demonstrate that bacterial Ku binds the DNA ends in a cooperative manner for synapsis initiation and remains stably bound at DNA junctions for several hours after ligation is completed, indicating that a system for removal of the proteins is active in vivo. Together these experiments shed light on the dynamics of bacterial NHEJ in DNA end recognition and processing. We speculate on the evolutionary similarities between bacterial and eukaryotic NHEJ and discuss how an increased understanding of bacterial NHEJ can open the door for future antibiotic therapies targeting this mechanism.

scholarly journals Role of the Mre11 Complex in Preserving Genome Integrity

Genes ◽  
2018 ◽  
Vol 9 (12) ◽  
pp. 589 ◽  
Author(s):  
Julyun Oh ◽  
Lorraine Symington
Keyword(s):  
Cell Cycle Progression ◽  
Genome Integrity ◽  
Replication Forks ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Atm Kinase ◽  
Strand Breaks ◽  
Mre11 Complex ◽  
Non Homologous End Joining ◽  
Dna Ends

DNA double-strand breaks (DSBs) are hazardous lesions that threaten genome integrity and cell survival. The DNA damage response (DDR) safeguards the genome by sensing DSBs, halting cell cycle progression and promoting repair through either non-homologous end joining (NHEJ) or homologous recombination (HR). The Mre11-Rad50-Xrs2/Nbs1 (MRX/N) complex is central to the DDR through its structural, enzymatic, and signaling roles. The complex tethers DNA ends, activates the Tel1/ATM kinase, resolves protein-bound or hairpin-capped DNA ends, and maintains telomere homeostasis. In addition to its role at DSBs, MRX/N associates with unperturbed replication forks, as well as stalled replication forks, to ensure complete DNA synthesis and to prevent chromosome rearrangements. Here, we summarize the significant progress made in characterizing the MRX/N complex and its various activities in chromosome metabolism.

scholarly journals DROSHA is recruited to DNA damage sites by the MRN complex to promote non-homologous end-joining

2021 ◽  
pp. jcs.249706
Author(s):  
Matteo Cabrini ◽  
Marco Roncador ◽  
Alessandro Galbiati ◽  
Lina Cipolla ◽  
Antonio Maffia ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Mrn Complex ◽  
Break Site ◽  
Non Homologous End Joining ◽  
Dna Ends

The DNA damage response (DDR) is the signaling cascade that recognizes DNA double-strand breaks (DSB) and promotes their resolution via the DNA repair pathways of Non-Homologous End Joining (NHEJ) or Homologous Recombination (HR). We and others have shown that DDR activation requires DROSHA. However, whether DROSHA exerts its functions by associating with damage sites, what controls its recruitment and how DROSHA influences DNA repair, remains poorly understood. Here we show that DROSHA associates to DSBs independently from transcription. Neither H2AX, nor ATM nor DNA-PK kinase activities are required for its recruitment to break site. Rather, DROSHA interacts with RAD50 and inhibition of MRN by Mirin treatment abolishes this interaction. MRN inactivation by RAD50 knockdown or mirin treatment prevents DROSHA recruitment to DSB and, as a consequence, also 53BP1 recruitment. During DNA repair, DROSHA inactivation reduces NHEJ and boosts HR frequency. Indeed, DROSHA knockdown also increase the association of downstream HR factors such as RAD51 to DNA ends. Overall, our results demonstrate that DROSHA is recruited at DSBs by the MRN complex and direct DNA repair toward NHEJ.

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