DNA End Resection: Mechanism and Control

2021 ◽  
Vol 55 (1) ◽  
pp. 285-307
Author(s):  
Petr Cejka ◽  
Lorraine S. Symington
Keyword(s):  
Double Strand Breaks ◽  
Nonhomologous End Joining ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Critical Determinant ◽  
Strand Breaks ◽  
Dna End Resection ◽  
And Control ◽  
End Resection ◽  
Dna Ends

DNA double-strand breaks (DSBs) are cytotoxic lesions that threaten genome integrity and cell viability. Typically, cells repair DSBs by either nonhomologous end joining (NHEJ) or homologous recombination (HR). The relative use of these two pathways depends on many factors, including cell cycle stage and the nature of the DNA ends. A critical determinant of repair pathway selection is the initiation of 5′→3′ nucleolytic degradation of DNA ends, a process referred to as DNA end resection. End resection is essential to create single-stranded DNA overhangs, which serve as the substrate for the Rad51 recombinase to initiate HR and are refractory to NHEJ repair. Here, we review recent insights into the mechanisms of end resection, how it is regulated, and the pathological consequences of its dysregulation.

Repair of DNA Double-Strand Breaks by the Nonhomologous End Joining Pathway

2021 ◽  
Vol 90 (1) ◽  
Author(s):  
Benjamin M. Stinson ◽  
Joseph J. Loparo
Keyword(s):  
Genome Stability ◽  
Double Strand Breaks ◽  
Nonhomologous End Joining ◽  
Annual Review ◽  
Publication Date ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Break Site ◽  
Dna Ends

DNA double-strand breaks pose a serious threat to genome stability. In vertebrates, these breaks are predominantly repaired by nonhomologous end joining (NHEJ), which pairs DNA ends in a multiprotein synaptic complex to promote their direct ligation. NHEJ is a highly versatile pathway that uses an array of processing enzymes to modify damaged DNA ends and enable their ligation. The mechanisms of end synapsis and end processing have important implications for genome stability. Rapid and stable synapsis is necessary to limit chromosome translocations that result from the mispairing of DNA ends. Furthermore, end processing must be tightly regulated to minimize mutations at the break site. Here, we review our current mechanistic understanding of vertebrate NHEJ, with a particular focus on end synapsis and processing. Expected final online publication date for the Annual Review of Biochemistry, Volume 90 is June 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

scholarly journals Antagonistic relationship of NuA4 with the Non-Homologous End-Joining machinery at DNA damage sites

2021 ◽  
Author(s):  
Salar Ahmad ◽  
Valerie Côté ◽  
Xue Cheng ◽  
Gaëlle Bourriquen ◽  
Vasileia Sapountzi ◽  
...  
Keyword(s):  
Budding Yeast ◽  
Double Strand Breaks ◽  
Repair Pathway ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Dna End Resection ◽  
Non Homologous End Joining ◽  
Relationship Of ◽  
End Resection

AbstractThe NuA4 histone acetyltransferase complex, apart from its known role in gene regulation, has also been directly implicated in the repair of DNA double-strand breaks (DSBs), favoring homologous recombination (HR) in S/G2 during the cell cycle. Here, we investigate the antagonistic relationship of NuA4 with non-homologous end joining (NHEJ) factors. We show that budding yeast Rad9, the 53BP1 ortholog, can inhibit NuA4 acetyltransferase activity when bound to chromatin in vitro. While we previously reported that NuA4 is recruited at DSBs during the S/G2 phase, we can also detect its recruitment in G1 when genes for NHEJ factors Rad9, Yku80 and Nej1 are mutated. This is accompanied with the binding of single-strand DNA binding protein RPA and Rad52, indicating DNA end resection in G1 as well as recruitment of the HR machinery. This NuA4 recruitment to DSBs in G1 depends on both Xrs2 and Lcd1/Ddc2. Introducing an acetyltransferase defective allele in these NHEJ mutant backgrounds decreases their hyper-resection phenotype in G1. Interestingly, we identified two novel non-histone acetylation targets of NuA4, Nej1 and Yku80. Acetyl-mimicking mutant of Nej1 inhibits repair of DNA breaks by NHEJ, decreases its interaction with other core NHEJ factors such as Yku80 and Lif1 and favors end resection. Altogether, these results establish a strong reciprocal antagonistic regulatory function of NuA4 and NHEJ factors in repair pathway choice and suggests a role of NuA4 in alternative repair mechanism that involves DNA-end resection in G1.Author SummaryDNA double-strand breaks (DSBs) are one of the most harmful form of DNA damage. Cells employ two major repair pathways to resolve DSBs: Homologous Recombination (HR) and Non-Homologous End Joining (NHEJ). Here we wanted to dissect further the role played by the NuA4 (Nucleosome acetyltransferase of histone H4) complex in the repair of DSBs. Budding yeast NuA4 complex, like its mammalian homolog TIP60 complex, has been shown to favor repair by HR. Here, we show that indeed budding yeast NuA4 and components of the NHEJ repair pathway share an antagonistic relationship. Deletion of NHEJ components enables increased recruitment of NuA4 in the vicinity of DSBs, where NuA4 favors the end resection process which is an underlying mechanism for HR repair. We also describe two independent modes responsible for the recruitment of NuA4 to DSB sites. Additionally, we also present two NHEJ core components as new targets of NuA4 acetyltransferase activity and suggest that these acetylation events can disassemble the NHEJ repair complex from DSBs, favoring repair by HR. Our study demonstrates the importance of NuA4 in the modulation of DSB repair pathway choice.

Repair pathway choice for double-strand breaks

2020 ◽  
Vol 64 (5) ◽  
pp. 765-777 ◽  
Author(s):  
Yixi Xu ◽  
Dongyi Xu
Keyword(s):  
Cell Cycle ◽  
Double Strand Breaks ◽  
Homologous Region ◽  
Gene Repair ◽  
Repair Pathway ◽  
Dna Double Strand Breaks ◽  
Environmental Radiation ◽  
Strand Breaks ◽  
Dna End Resection ◽  
End Resection

Abstract Deoxyribonucleic acid (DNA) is at a constant risk of damage from endogenous substances, environmental radiation, and chemical stressors. DNA double-strand breaks (DSBs) pose a significant threat to genomic integrity and cell survival. There are two major pathways for DSB repair: nonhomologous end-joining (NHEJ) and homologous recombination (HR). The extent of DNA end resection, which determines the length of the 3′ single-stranded DNA (ssDNA) overhang, is the primary factor that determines whether repair is carried out via NHEJ or HR. NHEJ, which does not require a 3′ ssDNA tail, occurs throughout the cell cycle. 53BP1 and the cofactors PTIP or RIF1-shieldin protect the broken DNA end, inhibit long-range end resection and thus promote NHEJ. In contrast, HR mainly occurs during the S/G2 phase and requires DNA end processing to create a 3′ tail that can invade a homologous region, ensuring faithful gene repair. BRCA1 and the cofactors CtIP, EXO1, BLM/DNA2, and the MRE11–RAD50–NBS1 (MRN) complex promote DNA end resection and thus HR. DNA resection is influenced by the cell cycle, the chromatin environment, and the complexity of the DNA end break. Herein, we summarize the key factors involved in repair pathway selection for DSBs and discuss recent related publications.

scholarly journals Autophosphorylation of DNA-PKCS regulates its dynamics at DNA double-strand breaks

2007 ◽  
Vol 177 (2) ◽  
pp. 219-229 ◽  
Author(s):  
Naoya Uematsu ◽  
Eric Weterings ◽  
Ken-ichi Yano ◽  
Keiko Morotomi-Yano ◽  
Burkhard Jakob ◽  
...  
Keyword(s):  
Kinase Activity ◽  
Laser System ◽  
Double Strand Breaks ◽  
Dependent Manner ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Dsb Repair ◽  
Strand Breaks ◽  
Dna Ends

The DNA-dependent protein kinase catalytic subunit (DNA-PKCS) plays an important role during the repair of DNA double-strand breaks (DSBs). It is recruited to DNA ends in the early stages of the nonhomologous end-joining (NHEJ) process, which mediates DSB repair. To study DNA-PKCS recruitment in vivo, we used a laser system to introduce DSBs in a specified region of the cell nucleus. We show that DNA-PKCS accumulates at DSB sites in a Ku80-dependent manner, and that neither the kinase activity nor the phosphorylation status of DNA-PKCS influences its initial accumulation. However, impairment of both of these functions results in deficient DSB repair and the maintained presence of DNA-PKCS at unrepaired DSBs. The use of photobleaching techniques allowed us to determine that the kinase activity and phosphorylation status of DNA-PKCS influence the stability of its binding to DNA ends. We suggest a model in which DNA-PKCS phosphorylation/autophosphorylation facilitates NHEJ by destabilizing the interaction of DNA-PKCS with the DNA ends.

scholarly journals Rejoining of DNA Double-Strand Breaks as a Function of Overhang Length

2005 ◽  
Vol 25 (3) ◽  
pp. 896-906 ◽  
Author(s):  
James M. Daley ◽  
Thomas E. Wilson
Keyword(s):  
Gc Content ◽  
Double Strand Breaks ◽  
Nonhomologous End Joining ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Single Strand Annealing ◽  
Primary Determinant ◽  
Overhang Length ◽  
Strand Annealing

ABSTRACT The ends of spontaneously occurring double-strand breaks (DSBs) may contain various lengths of single-stranded DNA, blocking lesions, and gaps and flaps generated by end annealing. To investigate the processing of such structures, we developed an assay in which annealed oligonucleotides are ligated onto the ends of a linearized plasmid which is then transformed into Saccharomyces cerevisiae. Reconstitution of a marker occurs only when the oligonucleotides are incorporated and repair is in frame, permitting rapid analysis of complex DSB ends. Here, we created DSBs with compatible overhangs of various lengths and asked which pathways are required for their precise repair. Three mechanisms of rejoining were observed, regardless of overhang polarity: nonhomologous end joining (NHEJ), a Rad52-dependent single-strand annealing-like pathway, and a third mechanism independent of the first two mechanisms. DSBs with overhangs of less than 4 bases were mainly repaired by NHEJ. Repair became less dependent on NHEJ when the overhangs were longer or had a higher GC content. Repair of overhangs greater than 8 nucleotides was as much as 150-fold more efficient, impaired 10-fold by rad52 mutation, and highly accurate. Reducing the microhomology extent between long overhangs reduced their repair dramatically, to less than NHEJ of comparable short overhangs. These data support a model in which annealing energy is a primary determinant of the rejoining efficiency and mechanism.

scholarly journals Histone chaperone Anp32e removes H2A.Z from DNA double-strand breaks and promotes nucleosome reorganization and DNA repair

2015 ◽  
Vol 112 (24) ◽  
pp. 7507-7512 ◽  
Author(s):  
Ozge Gursoy-Yuzugullu ◽  
Marina K. Ayrapetov ◽  
Brendan D. Price
Keyword(s):  
Dna Damage ◽  
Double Strand Breaks ◽  
Histone H4 ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Dsb Repair ◽  
Strand Breaks ◽  
Nucleosome Dynamics ◽  
Chromatin Template ◽  
End Resection

The repair of DNA double-strand breaks (DSBs) requires open, flexible chromatin domains. The NuA4–Tip60 complex creates these flexible chromatin structures by exchanging histone H2A.Z onto nucleosomes and promoting acetylation of histone H4. Here, we demonstrate that the accumulation of H2A.Z on nucleosomes at DSBs is transient, and that rapid eviction of H2A.Z is required for DSB repair. Anp32e, an H2A.Z chaperone that interacts with the C-terminal docking domain of H2A.Z, is rapidly recruited to DSBs. Anp32e functions to remove H2A.Z from nucleosomes, so that H2A.Z levels return to basal within 10 min of DNA damage. Further, H2A.Z removal by Anp32e disrupts inhibitory interactions between the histone H4 tail and the nucleosome surface, facilitating increased acetylation of histone H4 following DNA damage. When H2A.Z removal by Anp32e is blocked, nucleosomes at DSBs retain elevated levels of H2A.Z, and assume a more stable, hypoacetylated conformation. Further, loss of Anp32e leads to increased CtIP-dependent end resection, accumulation of single-stranded DNA, and an increase in repair by the alternative nonhomologous end joining pathway. Exchange of H2A.Z onto the chromatin and subsequent rapid removal by Anp32e are therefore critical for creating open, acetylated nucleosome structures and for controlling end resection by CtIP. Dynamic modulation of H2A.Z exchange and removal by Anp32e reveals the importance of the nucleosome surface and nucleosome dynamics in processing the damaged chromatin template during DSB repair.

scholarly journals CTCF cooperates with CtIP to drive homologous recombination repair of double-strand breaks

2019 ◽  
Vol 47 (17) ◽  
pp. 9160-9179 ◽  
Author(s):  
Soon Young Hwang ◽  
Mi Ae Kang ◽  
Chul Joon Baik ◽  
Yejin Lee ◽  
Ngo Thanh Hang ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Critical Role ◽  
Control Point ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
C Terminus ◽  
Dna End Resection ◽  
End Resection

Abstract The pleiotropic CCCTC-binding factor (CTCF) plays a role in homologous recombination (HR) repair of DNA double-strand breaks (DSBs). However, the precise mechanistic role of CTCF in HR remains largely unclear. Here, we show that CTCF engages in DNA end resection, which is the initial, crucial step in HR, through its interactions with MRE11 and CtIP. Depletion of CTCF profoundly impairs HR and attenuates CtIP recruitment at DSBs. CTCF physically interacts with MRE11 and CtIP and promotes CtIP recruitment to sites of DNA damage. Subsequently, CTCF facilitates DNA end resection to allow HR, in conjunction with MRE11–CtIP. Notably, the zinc finger domain of CTCF binds to both MRE11 and CtIP and enables proficient CtIP recruitment, DNA end resection and HR. The N-terminus of CTCF is able to bind to only MRE11 and its C-terminus is incapable of binding to MRE11 and CtIP, thereby resulting in compromised CtIP recruitment, DSB resection and HR. Overall, this suggests an important function of CTCF in DNA end resection through the recruitment of CtIP at DSBs. Collectively, our findings identify a critical role of CTCF at the first control point in selecting the HR repair pathway.

scholarly journals The internal region of CtIP negatively regulates DNA end resection

2020 ◽  
Vol 48 (10) ◽  
pp. 5485-5498 ◽  
Author(s):  
Sean Michael Howard ◽  
Ilaria Ceppi ◽  
Roopesh Anand ◽  
Roger Geiger ◽  
Petr Cejka
Keyword(s):  
Homologous Recombination ◽  
Regulatory Function ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Internal Region ◽  
Dna End Resection ◽  
End Resection

Abstract DNA double-strand breaks are repaired by end-joining or homologous recombination. A key-committing step of recombination is DNA end resection. In resection, phosphorylated CtIP first promotes the endonuclease of MRE11–RAD50–NBS1 (MRN). Subsequently, CtIP also stimulates the WRN/BLM–DNA2 pathway, coordinating thus both short and long-range resection. The structure of CtIP differs from its orthologues in yeast, as it contains a large internal unstructured region. Here, we conducted a domain analysis of CtIP to define the function of the internal region in DNA end resection. We found that residues 350–600 were entirely dispensable for resection in vitro. A mutant lacking these residues was unexpectedly more efficient than full-length CtIP in DNA end resection and homologous recombination in vivo, and consequently conferred resistance to lesions induced by the topoisomerase poison camptothecin, which require high MRN–CtIP-dependent resection activity for repair. This suggested that the internal CtIP region, further mapped to residues 550–600, may mediate a negative regulatory function to prevent over resection in vivo. The CtIP internal deletion mutant exhibited sensitivity to other DNA-damaging drugs, showing that upregulated resection may be instead toxic under different conditions. These experiments together identify a region within the central CtIP domain that negatively regulates DNA end resection.

scholarly journals Unexpected behavior of DNA polymerase Mu opposite template 8-oxo-7,8-dihydro-2′-guanosine

2019 ◽  
Vol 47 (17) ◽  
pp. 9410-9422 ◽  
Author(s):  
Andrea M Kaminski ◽  
Kishore K Chiruvella ◽  
Dale A Ramsden ◽  
Thomas A Kunkel ◽  
Katarzyna Bebenek ◽  
...  
Keyword(s):  
Double Strand Breaks ◽  
Nonhomologous End Joining ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Steady State Kinetics ◽  
Ligase Iv ◽  
Template Dna ◽  
Dna Substrate

Abstract DNA double-strand breaks (DSBs) resulting from reactive oxygen species generated by exposure to UV and ionizing radiation are characterized by clusters of lesions near break sites. Such complex DSBs are repaired slowly, and their persistence can have severe consequences for human health. We have therefore probed DNA break repair containing a template 8-oxo-7,8-dihydro-2′-guanosine (8OG) by Family X Polymerase μ (Pol μ) in steady-state kinetics and cell-based assays. Pol μ tolerates 8OG-containing template DNA substrates, and the filled products can be subsequently ligated by DNA Ligase IV during Nonhomologous end-joining. Furthermore, Pol μ exhibits a strong preference for mutagenic bypass of 8OG by insertion of adenine. Crystal structures reveal that the template 8OG is accommodated in the Pol μ active site with none of the DNA substrate distortions observed for Family X siblings Pols β or λ. Kinetic characterization of template 8OG bypass indicates that Pol μ inserts adenosine nucleotides with weak sugar selectivity and, given the high cellular concentration of ATP, likely performs its role in repair of complex 8OG-containing DSBs using ribonucleotides.

scholarly journals Involvement of Artemis in nonhomologous end-joining during immunoglobulin class switch recombination

2008 ◽  
Vol 205 (13) ◽  
pp. 3031-3040 ◽  
Author(s):  
Likun Du ◽  
Mirjam van der Burg ◽  
Sergey W. Popov ◽  
Ashwin Kotnis ◽  
Jacques J.M. van Dongen ◽  
...  
Keyword(s):  
Class Switch Recombination ◽  
Double Strand Breaks ◽  
Nonhomologous End Joining ◽  
Igg Subclass ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Immunoglobulin Class ◽  
Class Switch ◽  
Human B Cells

DNA double-strand breaks (DSBs) introduced in the switch (S) regions are intermediates during immunoglobulin class switch recombination (CSR). These breaks are subsequently recognized, processed, and joined, leading to recombination of the two S regions. Nonhomologous end-joining (NHEJ) is believed to be the principle mechanism involved in DSB repair during CSR. One important component in NHEJ, Artemis, has however been considered to be dispensable for efficient CSR. In this study, we have characterized the S recombinational junctions from Artemis-deficient human B cells. Sμ–Sα junctions could be amplified from all patients tested and were characterized by a complete lack of “direct” end-joining and a remarkable shift in the use of an alternative, microhomology-based end-joining pathway. Sμ–Sγ junctions could only be amplified from one patient who carries “hypomorphic” mutations. Although these Sμ–Sγ junctions appear to be normal, a significant increase of an unusual type of sequential switching from immunoglobulin (Ig)M, through one IgG subclass, to a different IgG subclass was observed, and the Sγ–Sγ junctions showed long microhomologies. Thus, when the function of Artemis is impaired, varying modes of CSR junction resolution may be used for different S regions. Our findings strongly link Artemis to the predominant NHEJ pathway during CSR.

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