scholarly journals Phosphorylated CtIP bridges DNA to promote annealing of broken ends

2020 ◽  
Vol 117 (35) ◽  
pp. 21403-21412
Author(s):  
Robin Öz ◽  
Sean M. Howard ◽  
Rajhans Sharma ◽  
Hanna Törnkvist ◽  
Ilaria Ceppi ◽  
...  
Keyword(s):  
Nuclease Activity ◽  
Strand Break ◽  
End Joining ◽  
Oligomeric State ◽  
Mrn Complex ◽  
Nanofluidic Channels ◽  
Structural Functions ◽  
Dna Double Strand Break ◽  
Yeast Homolog ◽  
Dna Ends

The early steps of DNA double-strand break (DSB) repair in human cells involve the MRE11-RAD50-NBS1 (MRN) complex and its cofactor, phosphorylated CtIP. The roles of these proteins in nucleolytic DSB resection are well characterized, but their role in bridging the DNA ends for efficient and correct repair is much less explored. Here we study the binding of phosphorylated CtIP, which promotes the endonuclease activity of MRN, to single long (∼50 kb) DNA molecules using nanofluidic channels and compare it to the yeast homolog Sae2. CtIP bridges DNA in a manner that depends on the oligomeric state of the protein, and truncated mutants demonstrate that the bridging depends on CtIP regions distinct from those that stimulate the nuclease activity of MRN. Sae2 is a much smaller protein than CtIP, and its bridging is significantly less efficient. Our results demonstrate that the nuclease cofactor and structural functions of CtIP may depend on the same protein population, which may be crucial for CtIP functions in both homologous recombination and microhomology-mediated end-joining.

scholarly journals XRCC4 and XLF form long helical protein filaments suitable for DNA end protection and alignment to facilitate DNA double strand break repair

2013 ◽  
Vol 91 (1) ◽  
pp. 31-41 ◽  
Author(s):  
Brandi L. Mahaney ◽  
Michal Hammel ◽  
Katheryn Meek ◽  
John A. Tainer ◽  
Susan P. Lees-Miller
Keyword(s):  
Strand Break ◽  
Nonhomologous End Joining ◽  
Scaffolding Protein ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Dna Double Strand Break ◽  
Helical Protein ◽  
Gene 4 ◽  
Dna Ends

DNA double strand breaks (DSBs), induced by ionizing radiation (IR) and endogenous stress including replication failure, are the most cytotoxic form of DNA damage. In human cells, most IR-induced DSBs are repaired by the nonhomologous end joining (NHEJ) pathway. One of the most critical steps in NHEJ is ligation of DNA ends by DNA ligase IV (LIG4), which interacts with, and is stabilized by, the scaffolding protein X-ray cross-complementing gene 4 (XRCC4). XRCC4 also interacts with XRCC4-like factor (XLF, also called Cernunnos); yet, XLF has been one of the least mechanistically understood proteins and precisely how XLF functions in NHEJ has been enigmatic. Here, we examine current combined structural and mutational findings that uncover integrated functions of XRCC4 and XLF and reveal their interactions to form long, helical protein filaments suitable to protect and align DSB ends. XLF–XRCC4 provides a global structural scaffold for ligating DSBs without requiring long DNA ends, thus ensuring accurate and efficient ligation and repair. The assembly of these XRCC4–XLF filaments, providing both DNA end protection and alignment, may commit cells to NHEJ with general biological implications for NHEJ and DSB repair processes and their links to cancer predispositions and interventions.

scholarly journals DNA End Joining: G0-ing to the Core

Biomolecules ◽  
2021 ◽  
Vol 11 (10) ◽  
pp. 1487
Author(s):  
Richard L. Frock ◽  
Cheyenne Sadeghi ◽  
Jodie Meng ◽  
Jing L. Wang
Keyword(s):  
Strand Break ◽  
End Joining ◽  
The Core ◽  
Double Stranded Dna ◽  
Random Pairing ◽  
Dna Double Strand Break ◽  
Damage Response ◽  
Alternative End Joining ◽  
Non Homologous End Joining ◽  
Dna Ends

Humans have evolved a series of DNA double-strand break (DSB) repair pathways to efficiently and accurately rejoin nascently formed pairs of double-stranded DNA ends (DSEs). In G0/G1-phase cells, non-homologous end joining (NHEJ) and alternative end joining (A-EJ) operate to support covalent rejoining of DSEs. While NHEJ is predominantly utilized and collaborates extensively with the DNA damage response (DDR) to support pairing of DSEs, much less is known about A-EJ collaboration with DDR factors when NHEJ is absent. Non-cycling lymphocyte progenitor cells use NHEJ to complete V(D)J recombination of antigen receptor genes, initiated by the RAG1/2 endonuclease which holds its pair of targeted DSBs in a synapse until each specified pair of DSEs is handed off to the NHEJ DSB sensor complex, Ku. Similar to designer endonuclease DSBs, the absence of Ku allows for A-EJ to access RAG1/2 DSEs but with random pairing to complete their repair. Here, we describe recent insights into the major phases of DSB end joining, with an emphasis on synapsis and tethering mechanisms, and bring together new and old concepts of NHEJ vs. A-EJ and on RAG2-mediated repair pathway choice.

scholarly journals Nej1(XLF) inhibits Sae2(CTIP)-Dna2(DNA2) mediated resection at DNA double strand breaks

2021 ◽  
Author(s):  
Aditya Mojumdar ◽  
Nancy Adam ◽  
Jennifer A Cobb
Keyword(s):  
Synthetic Lethality ◽  
Nuclease Activity ◽  
Strand Break ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Genomic Deletions ◽  
Dna Double Strand Break ◽  
Break Site ◽  
Dna End Resection ◽  
Non Homologous End Joining

The two major pathways of DNA double strand break (DSB) repair, non-homologous end-joining (NHEJ) and homologous recombination (HR), are highly conserved from yeast to mammals. Regulated 5 DNA end resection is important for repair pathway choice and repair outcomes. Nej1 was first identified as a canonical NHEJ factor involved in stimulating the ligation of broken DNA ends and, more recently, it was shown to be important for DNA end-bridging and inhibiting Dna2-Sgs1 mediated 5 resection. Dna2 localizes to DSBs in the absence of Sgs1 through interactions with Mre11 and Sae2 and DNA damage sensitivity is greater in cells lacking Dna2 nuclease activity compared to sgs1∆ mutants. Dna2-Sae2 mediated 5 resection is down-regulated by Nej1, which itself interacts with Sae2. The resection defect of sae2∆ and the synthetic lethality of sae2∆ sgs1∆ are reversed by deletion of NEJ1 and dependent on Dna2 nuclease activity. Our work demonstrates the importance of Nej1 in inhibiting short-range resection at a DSB by Dna2-Sae2, a critical regulatory mechanism that prevents the formation of genomic deletions at the break site.

scholarly journals DNA-dependent protein kinase promotes DNA end processing by MRN and CtIP

2020 ◽  
Vol 6 (2) ◽  
pp. eaay0922 ◽  
Author(s):  
Rajashree A. Deshpande ◽  
Logan R. Myler ◽  
Michael M. Soniat ◽  
Nodar Makharashvili ◽  
Linda Lee ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Homologous Recombination ◽  
Single Molecule ◽  
Strand Break ◽  
Nonhomologous End Joining ◽  
End Joining ◽  
Dependent Protein Kinase ◽  
Mrn Complex ◽  
Dependent Protein ◽  
Dna Ends

The repair of DNA double-strand breaks occurs through nonhomologous end joining or homologous recombination in vertebrate cells—a choice that is thought to be decided by a competition between DNA-dependent protein kinase (DNA-PK) and the Mre11/Rad50/Nbs1 (MRN) complex but is not well understood. Using ensemble biochemistry and single-molecule approaches, here, we show that the MRN complex is dependent on DNA-PK and phosphorylated CtIP to perform efficient processing and resection of DNA ends in physiological conditions, thus eliminating the competition model. Endonucleolytic removal of DNA-PK–bound DNA ends is also observed at double-strand break sites in human cells. The involvement of DNA-PK in MRN-mediated end processing promotes an efficient and sequential transition from nonhomologous end joining to homologous recombination by facilitating DNA-PK removal.

scholarly journals CRISPR/Cas9 cleavages in budding yeast reveal templated insertions and strand-specific insertion/deletion profiles

2018 ◽  
Vol 115 (9) ◽  
pp. E2040-E2047 ◽  
Author(s):  
Brenda R. Lemos ◽  
Adam C. Kaplan ◽  
Ji Eun Bae ◽  
Alexander E. Ferrazzoli ◽  
James Kuo ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Mammalian Cells ◽  
Budding Yeast ◽  
Nuclease Activity ◽  
Strand Break ◽  
End Joining ◽  
Guide Rna ◽  
Dna Double Strand Break ◽  
Nhej Repair ◽  
Dna Strands

Harnessing CRISPR-Cas9 technology provides an unprecedented ability to modify genomic loci via DNA double-strand break (DSB) induction and repair. We analyzed nonhomologous end-joining (NHEJ) repair induced by Cas9 in budding yeast and found that the orientation of binding of Cas9 and its guide RNA (gRNA) profoundly influences the pattern of insertion/deletions (indels) at the site of cleavage. A common indel created by Cas9 is a 1-bp (+1) insertion that appears to result from Cas9 creating a 1-nt 5′ overhang that is filled in by a DNA polymerase and ligated. The origin of +1 insertions was investigated by using two gRNAs with PAM sequences located on opposite DNA strands but designed to cleave the same sequence. These templated +1 insertions are dependent on the X-family DNA polymerase, Pol4. Deleting Pol4 also eliminated +2 and +3 insertions, which are biased toward homonucleotide insertions. Using inverted PAM sequences, we also found significant differences in overall NHEJ efficiency and repair profiles, suggesting that the binding of the Cas9:gRNA complex influences subsequent NHEJ processing. As with events induced by the site-specific HO endonuclease, CRISPR-Cas9–mediated NHEJ repair depends on the Ku heterodimer and DNA ligase 4. Cas9 events are highly dependent on the Mre11-Rad50-Xrs2 complex, independent of Mre11’s nuclease activity. Inspection of the outcomes of a large number of Cas9 cleavage events in mammalian cells reveals a similar templated origin of +1 insertions in human cells, but also a significant frequency of similarly templated +2 insertions.

scholarly journals A noncatalytic function of the ligation complex during nonhomologous end joining

2013 ◽  
Vol 200 (2) ◽  
pp. 173-186 ◽  
Author(s):  
Jessica Cottarel ◽  
Philippe Frit ◽  
Oriane Bombarde ◽  
Bernard Salles ◽  
Aurélie Négrel ◽  
...  
Keyword(s):  
Ectopic Expression ◽  
Strand Break ◽  
Nonhomologous End Joining ◽  
End Joining ◽  
Free System ◽  
Cell Extracts ◽  
Dna Double Strand Break ◽  
Dependent Protein ◽  
A Cell ◽  
Dna Ends

Nonhomologous end joining is the primary deoxyribonucleic acid (DNA) double-strand break repair pathway in multicellular eukaryotes. To initiate repair, Ku binds DNA ends and recruits the DNA-dependent protein kinase (DNA-PK) catalytic subunit (DNA-PKcs) forming the holoenzyme. Early end synapsis is associated with kinase autophosphorylation. The XRCC4 (X4)–DNA Ligase IV (LIG4) complex (X4LIG4) executes the final ligation promoted by Cernunnos (Cer)–X4-like factor (XLF). In this paper, using a cell-free system that recapitulates end synapsis and DNA-PKcs autophosphorylation, we found a defect in both activities in human cell extracts lacking LIG4. LIG4 also stimulated the DNA-PKcs autophosphorylation in a reconstitution assay with purified components. We additionally uncovered a kinase autophosphorylation defect in LIG4-defective cells that was corrected by ectopic expression of catalytically dead LIG4. Finally, our data support a contribution of Cer-XLF to this unexpected early role of the ligation complex in end joining. We propose that productive end joining occurs by early formation of a supramolecular entity containing both DNA-PK and X4LIG4–Cer-XLF complexes on DNA ends.

scholarly journals CRISPR/Cas9 cleavages in budding yeast reveal templated insertions and strand-specific insertion/deletion profiles

2017 ◽  
Author(s):  
Brenda R. Lemos ◽  
Adam C. Kaplan ◽  
Ji Eun Bae ◽  
Alex Ferazzoli ◽  
James Kuo ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Mammalian Cells ◽  
Budding Yeast ◽  
Nuclease Activity ◽  
Strand Break ◽  
End Joining ◽  
Guide Rna ◽  
Dna Double Strand Break ◽  
Nhej Repair ◽  
Dna Strands

AbstractHarnessing CRISPR-Cas9 technology has provided an unprecedented ability to modify genomic loci via DNA double-strand break (DSB) induction and repair. We have analyzed nonhomologous end-joining (NHEJ) repair induced by Cas9 in the budding yeast Saccharomyces cerevisiae and find that the orientation of binding of Cas9 and its guide RNA (gRNA) profoundly influences the pattern of insertion/deletions (indels) at the site of cleavage. A common indel created by Cas9 is a one base pair (+1) insertion that appears to result from Cas9 creating a 1-bp 5’ overhang that is filled in by a DNA polymerase and ligated. The origin of +1 insertions was investigated by using two gRNAs with PAM sequences located on opposite DNA strands but designed to cleave the same sequence. These templated +1 insertions are dependent on the X-family DNA polymerase, Pol4. Deleting Pol4 also eliminated +2 and +3 insertions, which were biased toward homonucleotide insertions. Using inverted PAM (iPAM) sequences, we also found significant differences in overall NHEJ efficiency and repair profiles, suggesting that the binding of the Cas9::gRNA complex influences subsequent NHEJ processing. As with well-studied events induced by the site-specific HO endonuclease, CRISPR-Cas9 mediated NHEJ repair depends on the Ku heterodimer and DNA ligase 4. Cas9 events, however, are highly dependent on the Mre11-Rad50-Xrs2 complex, independent of Mre11’s nuclease activity. Inspection of the outcomes of a large number of Cas9 cleavage events in mammalian cells (van Overbeek et al., 2016) reveals a similar templated origin of +1 insertions in human cells, but also a significant frequency of similarly templated +2 insertions.

scholarly journals The Transcriptional Histone Acetyltransferase Cofactor TRRAP Associates with the MRN Repair Complex and Plays a Role in DNA Double-Strand Break Repair

2006 ◽  
Vol 26 (2) ◽  
pp. 402-412 ◽  
Author(s):  
Flavie Robert ◽  
Sara Hardy ◽  
Zita Nagy ◽  
Céline Baldeyron ◽  
Rabih Murr ◽  
...  
Keyword(s):  
Cell Cycle Progression ◽  
Histone Acetyltransferase ◽  
Gel Filtration ◽  
Embryonic Stem ◽  
Strand Break ◽  
Mitotic Checkpoint ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Mrn Complex

ABSTRACT Transactivation-transformation domain-associated protein (TRRAP) is a component of several multiprotein histone acetyltransferase (HAT) complexes implicated in transcriptional regulation. TRRAP was shown to be required for the mitotic checkpoint and normal cell cycle progression. MRE11, RAD50, and NBS1 (product of the Nijmegan breakage syndrome gene) form the MRN complex that is involved in the detection, signaling, and repair of DNA double-strand breaks (DSBs). By using double immunopurification, mass spectrometry, and gel filtration, we describe the stable association of TRRAP with the MRN complex. The TRRAP-MRN complex is not associated with any detectable HAT activity, while the isolated other TRRAP complexes, containing either GCN5 or TIP60, are. TRRAP-depleted extracts show a reduced nonhomologous DNA end-joining activity in vitro. Importantly, small interfering RNA knockdown of TRRAP in HeLa cells or TRRAP knockout in mouse embryonic stem cells inhibit the DSB end-joining efficiency and the precise nonhomologous end-joining process, further suggesting a functional involvement of TRRAP in the DSB repair processes. Thus, TRRAP may function as a molecular link between DSB signaling, repair, and chromatin remodeling.

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