scholarly journals Organization and dynamics of the nonhomologous end-joining machinery during DNA double-strand break repair

2015 ◽  
Vol 112 (20) ◽  
pp. E2575-E2584 ◽  
Author(s):  
Dylan A. Reid ◽  
Sarah Keegan ◽  
Alejandra Leo-Macias ◽  
Go Watanabe ◽  
Natasha T. Strande ◽  
...  
Keyword(s):  
Single Molecule ◽  
Strand Break ◽  
Nonhomologous End Joining ◽  
Dna Ligase ◽  
End Joining ◽  
Dna Ligase Iv ◽  
Ligase Iv ◽  
End To End

Nonhomologous end-joining (NHEJ) is a major repair pathway for DNA double-strand breaks (DSBs), involving synapsis and ligation of the broken strands. We describe the use of in vivo and in vitro single-molecule methods to define the organization and interaction of NHEJ repair proteins at DSB ends. Super-resolution fluorescence microscopy allowed the precise visualization of XRCC4, XLF, and DNA ligase IV filaments adjacent to DSBs, which bridge the broken chromosome and direct rejoining. We show, by single-molecule FRET analysis of the Ku/XRCC4/XLF/DNA ligase IV NHEJ ligation complex, that end-to-end synapsis involves a dynamic positioning of the two ends relative to one another. Our observations form the basis of a new model for NHEJ that describes the mechanism whereby filament-forming proteins bridge DNA DSBs in vivo. In this scheme, the filaments at either end of the DSB interact dynamically to achieve optimal configuration and end-to-end positioning and ligation.

scholarly journals The C-Terminal Domain of Cernunnos/XLF Is Dispensable for DNA Repair In Vivo

2008 ◽  
Vol 29 (5) ◽  
pp. 1116-1122 ◽  
Author(s):  
Laurent Malivert ◽  
Isabelle Callebaut ◽  
Paola Rivera-Munoz ◽  
Alain Fischer ◽  
Jean-Paul Mornon ◽  
...  
Keyword(s):  
Dna Repair ◽  
Nonhomologous End Joining ◽  
Dna Ligase ◽  
Repair Pathway ◽  
End Joining ◽  
Functional Assays ◽  
The Core ◽  
Ligase Iv

ABSTRACT The core nonhomologous end-joining DNA repair pathway is composed of seven factors: Ku70, Ku80, DNA-PKcs, Artemis, XRCC4 (X4), DNA ligase IV (L4), and Cernunnos/XLF (Cernunnos). Although Cernunnos and X4 are structurally related and participate in the same complex together with L4, they have distinct functions during DNA repair. L4 relies on X4 but not on Cernunnos for its stability, and L4 is required for optimal interaction of Cernunnos with X4. We demonstrate here, using in vitro-generated Cernunnos mutants and a series of functional assays in vivo, that the C-terminal region of Cernunnos is dispensable for its activity during DNA repair.

scholarly journals Mutations of the Yku80 C Terminus and Xrs2 FHA Domain Specifically Block Yeast Nonhomologous End Joining

2005 ◽  
Vol 25 (24) ◽  
pp. 10782-10790 ◽  
Author(s):  
Phillip L. Palmbos ◽  
James M. Daley ◽  
Thomas E. Wilson
Keyword(s):  
Mutational Analysis ◽  
Strand Break ◽  
Nonhomologous End Joining ◽  
Dna Ligase ◽  
End Joining ◽  
Fha Domain ◽  
Dna Ligase Iv ◽  
C Terminus ◽  
Ligase Iv ◽  
Two Hybrid

ABSTRACT The nonhomologous end-joining (NHEJ) pathway of DNA double-strand break repair requires three protein complexes in Saccharomyces cerevisiae: MRX (Mre11-Rad50-Xrs2), Ku (Ku70-Ku80), and DNA ligase IV (Dnl4-Lif1-Nej1). Much is known about the interactions that mediate the formation of each complex, but little is known about how they act together during repair. A comprehensive yeast two-hybrid screen of the NHEJ factors of S. cerevisiae revealed all known interactions within the MRX, Ku, and DNA ligase IV complexes, as well as three additional, weaker interactions between Yku80-Dnl4, Xrs2-Lif1, and Mre11-Yku80. Individual and combined deletions of the Yku80 C terminus and the Xrs2 forkhead-associated (FHA) domain were designed based on the latter two-hybrid results. These deletions synergistically blocked NHEJ but not the telomere and recombination functions of Ku and MRX, confirming that these protein regions are functionally important specifically for NHEJ. Further mutational analysis of Yku80 identified a putative C-terminal amphipathic α-helix that is both required for its NHEJ function and strikingly similar to a DNA-dependent protein kinase interaction motif in human Ku80. These results identify a novel role in yeast NHEJ for the poorly characterized Ku80 C-terminal and Xrs2 FHA domains, and they suggest that redundant binding of DNA ligase IV facilitates completion of this DNA repair event.

scholarly journals DNA ligase IV mutations confer shorter lifespan and increased sensitivity to nutrient stress in Drosophila melanogaster

2021 ◽  
Author(s):  
Rashmi Joshi ◽  
Surya Jyoti Banerjee ◽  
Jennifer Curtiss ◽  
Amanda K. Ashley
Keyword(s):  
Drosophila Melanogaster ◽  
Strand Break ◽  
Nonhomologous End Joining ◽  
Dna Ligase ◽  
End Joining ◽  
Dna Ligase Iv ◽  
Dna Double Strand Break ◽  
Ligase Iv ◽  
Increased Sensitivity

AbstractThe nonhomologous end-joining pathway is a primary DNA double-strand break repair pathway in eukaryotes. DNA ligase IV (Lig4) catalyzes the final step of DNA end ligation in this pathway. Partial loss of Lig4 in mammals causes Lig4 syndrome, while complete loss is embryonically lethal. DNA ligase 4 (DNAlig4) null Drosophila melanogaster is viable, but sensitive to ionizing radiation during early development. We proposed to explore if DNAlig4 loss induced other long-term sensitivities and defects in D. melanogaster. We demonstrated that DNAlig4 mutant strains had decreased lifespan and lower resistance to nutrient deprivation, indicating Lig4 is required for maintaining health and longevity in D. melanogaster.

scholarly journals Structural Basis of DNA Ligase IV-Artemis Interaction in Nonhomologous End-Joining

Cell Reports ◽  
2012 ◽  
Vol 2 (6) ◽  
pp. 1505-1512 ◽  
Author(s):  
Pablo De Ioannes ◽  
Shruti Malu ◽  
Patricia Cortes ◽  
Aneel K. Aggarwal
Keyword(s):  
Nonhomologous End Joining ◽  
Structural Basis ◽  
Dna Ligase ◽  
End Joining ◽  
Dna Ligase Iv ◽  
Ligase Iv

scholarly journals Yeast DNA ligase IV mutations reveal a nonhomologous end joining function of BRCT1 distinct from XRCC4/Lif1 binding

DNA Repair ◽  
2014 ◽  
Vol 24 ◽  
pp. 37-45 ◽  
Author(s):  
Kishore K. Chiruvella ◽  
Brian M. Renard ◽  
Shanda R. Birkeland ◽  
Sham Sunder ◽  
Zhuobin Liang ◽  
...  
Keyword(s):  
Nonhomologous End Joining ◽  
Dna Ligase ◽  
End Joining ◽  
Dna Ligase Iv ◽  
Ligase Iv

scholarly journals XLF Interacts with the XRCC4-DNA Ligase IV Complex to Promote DNA Nonhomologous End-Joining

Cell ◽  
2006 ◽  
Vol 124 (2) ◽  
pp. 301-313 ◽  
Author(s):  
Peter Ahnesorg ◽  
Philippa Smith ◽  
Stephen P. Jackson
Keyword(s):  
Nonhomologous End Joining ◽  
Dna Ligase ◽  
End Joining ◽  
Dna Ligase Iv ◽  
Ligase Iv

scholarly journals DNA Ligase IV Guides End-Processing Choice during Nonhomologous End Joining

Cell Reports ◽  
2017 ◽  
Vol 20 (12) ◽  
pp. 2810-2819 ◽  
Author(s):  
Michael P. Conlin ◽  
Dylan A. Reid ◽  
George W. Small ◽  
Howard H. Chang ◽  
Go Watanabe ◽  
...  
Keyword(s):  
Nonhomologous End Joining ◽  
Dna Ligase ◽  
End Joining ◽  
Dna Ligase Iv ◽  
Ligase Iv

scholarly journals XRCC4/XLF Interaction Is Variably Required for DNA Repair and Is Not Required for Ligase IV Stimulation

2015 ◽  
Vol 35 (17) ◽  
pp. 3017-3028 ◽  
Author(s):  
Sunetra Roy ◽  
Abinadabe J. de Melo ◽  
Yao Xu ◽  
Satish K. Tadi ◽  
Aurélie Négrel ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Double Strand Breaks ◽  
Nonhomologous End Joining ◽  
Dna Ligase ◽  
End Joining ◽  
Strand Breaks ◽  
Ligase Iv ◽  
Cell Strains ◽  
Dna Ends

The classic nonhomologous end-joining (c-NHEJ) pathway is largely responsible for repairing double-strand breaks (DSBs) in mammalian cells. XLF stimulates the XRCC4/DNA ligase IV complex by an unknown mechanism. XLF interacts with XRCC4 to form filaments of alternating XRCC4 and XLF dimers that bridge DNA endsin vitro, providing a mechanism by which XLF might stimulate ligation. Here, we characterize two XLF mutants that do not interact with XRCC4 and cannot form filaments or bridge DNAin vitro. One mutant is fully sufficient in stimulating ligation by XRCC4/Lig4in vitro; the other is not. This separation-of-function mutant (which must function as an XLF homodimer) fully complements the c-NHEJ deficits of some XLF-deficient cell strains but not others, suggesting a variable requirement for XRCC4/XLF interaction in living cells. To determine whether the lack of XRCC4/XLF interaction (and potential bridging) can be compensated for by other factors, candidate repair factors were disrupted in XLF- or XRCC4-deficient cells. The loss of either ATM or the newly described XRCC4/XLF-like factor, PAXX, accentuates the requirement for XLF. However, in the case of ATM/XLF loss (but not PAXX/XLF loss), this reflects a greater requirement for XRCC4/XLF interaction.

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