Nonhomologous end-joining of site-specific but not of radiation-induced DNA double-strand breaks is reduced in the presence of wild-type p53

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.

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

Nucleic Acids Research ◽

10.1093/nar/gkz680 ◽

2019 ◽

Vol 47 (17) ◽

pp. 9410-9422 ◽

Cited By ~ 1

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.

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

Journal of Experimental Medicine ◽

10.1084/jem.20081915 ◽

2008 ◽

Vol 205 (13) ◽

pp. 3031-3040 ◽

Cited By ~ 34

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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Pluronic copolymer encapsulated SCR7 as a potential anticancer agent

Faraday Discussions ◽

10.1039/c4fd00176a ◽

2015 ◽

Vol 177 ◽

pp. 155-161 ◽

Cited By ~ 13

Author(s):

Franklin John ◽

Jinu George ◽

Mrinal Srivastava ◽

P. A. Hassan ◽

V. K. Aswal ◽

...

Keyword(s):

Therapeutic Agent ◽

Analytical Techniques ◽

Double Strand Breaks ◽

Nonhomologous End Joining ◽

Physical Interaction ◽

Dna Double Strand Breaks ◽

End Joining ◽

Strand Breaks ◽

Anticancer Properties

Nonhomologous end joining (NHEJ) of DNA double strand breaks (DSBs) inside cells can be selectively inhibited by 5,6-bis-(benzylideneamino)-2-mercaptopyrimidin-4-ol (SCR7) which possesses anticancer properties. The hydrophobicity of SCR7 decreases its bioavailability which is a major setback in the utilization of this compound as a therapeutic agent. In order to circumvent the drawback of SCR7, we prepared a polymer encapsulated form of SCR7. The physical interaction of SCR7 and Pluronic® copolymer is evident from different analytical techniques. The in vitro cytotoxicity of the drug formulations is established using the MTT assay.

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Loss of autophagy causes a synthetic lethal deficiency in DNA repair

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1409563112 ◽

2015 ◽

Vol 112 (3) ◽

pp. 773-778 ◽

Cited By ~ 75

Author(s):

Emma Y. Liu ◽

Naihan Xu ◽

Jim O’Prey ◽

Laurence Y. Lao ◽

Sanket Joshi ◽

...

Keyword(s):

Dna Repair ◽

Repair Process ◽

Double Strand Breaks ◽

Nonhomologous End Joining ◽

Cytoplasmic Process ◽

Dna Double Strand Breaks ◽

End Joining ◽

Synthetic Lethal ◽

Strand Breaks ◽

Genomic Damage

(Macro)autophagy delivers cellular constituents to lysosomes for degradation. Although a cytoplasmic process, autophagy-deficient cells accumulate genomic damage, but an explanation for this effect is currently unclear. We report here that inhibition of autophagy causes elevated proteasomal activity leading to enhanced degradation of checkpoint kinase 1 (Chk1), a pivotal factor for the error-free DNA repair process, homologous recombination (HR). We show that loss of autophagy critically impairs HR and that autophagy-deficient cells accrue micronuclei and sub-G1 DNA, indicators of diminished genomic integrity. Moreover, due to impaired HR, autophagy-deficient cells are hyperdependent on nonhomologous end joining (NHEJ) for repair of DNA double-strand breaks. Consequently, inhibition of NHEJ following DNA damage in the absence of autophagy results in persistence of genomic lesions and rapid cell death. Because autophagy deficiency occurs in several diseases, these findings constitute an important link between autophagy and DNA repair and highlight a synthetic lethal strategy to kill autophagy-deficient cells.

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Caffeine-Induced Radiosensitization is Independent of Nonhomologous End Joining of DNA Double-Strand Breaks

Radiation Research ◽

10.1667/0033-7587(2003)159[0426:ciriio]2.0.co;2 ◽

2003 ◽

Vol 159 (3) ◽

pp. 426-432 ◽

Cited By ~ 28

Author(s):

Xiang Wang ◽

Huichen Wang ◽

George Iliakis ◽

Ya Wang

Keyword(s):

Double Strand Breaks ◽

Nonhomologous End Joining ◽

Dna Double Strand Breaks ◽

End Joining ◽

Strand Breaks

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

Annual Review of Biochemistry ◽

10.1146/annurev-biochem-080320-110356 ◽

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.

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