Molecular basis of microhomology-mediated end-joining by purified full-length Polθ

Abstract DNA polymerase θ (Polθ) is a unique polymerase-helicase fusion protein that promotes microhomology-mediated end-joining (MMEJ) of DNA double-strand breaks (DSBs). How full-length human Polθ performs MMEJ at the molecular level remains unknown. Using a biochemical approach, we find that the helicase is essential for Polθ MMEJ of long ssDNA overhangs which model resected DSBs. Remarkably, Polθ MMEJ of ssDNA overhangs requires polymerase-helicase attachment, but not the disordered central domain, and occurs independently of helicase ATPase activity. Using single-particle microscopy and biophysical methods, we find that polymerase-helicase attachment promotes multimeric gel-like Polθ complexes that facilitate DNA accumulation, DNA synapsis, and MMEJ. We further find that the central domain regulates Polθ multimerization and governs its DNA substrate requirements for MMEJ. These studies identify unexpected functions for the helicase and central domain and demonstrate the importance of polymerase-helicase tethering in MMEJ and the structural organization of Polθ.

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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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Faculty Opinions recommendation of Polq-Mediated End Joining Is Essential for Surviving DNA Double-Strand Breaks during Early Zebrafish Development.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.726318081.793518723 ◽

2016 ◽

Author(s):

Cecilia Moens

Keyword(s):

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

End Joining ◽

Strand Breaks ◽

Zebrafish Development

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Analysis of chromatid-break-repair detects a homologous recombination to non-homologous end-joining switch with increasing load of DNA double-strand breaks

Mutation Research/Genetic Toxicology and Environmental Mutagenesis ◽

10.1016/j.mrgentox.2021.503372 ◽

2021 ◽

pp. 503372

Author(s):

Tamara Murmann-Konda ◽

Aashish Soni ◽

Martin Stuschke ◽

George Iliakis

Keyword(s):

Homologous Recombination ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

End Joining ◽

Strand Breaks ◽

Chromatid Break ◽

Break Repair ◽

Non Homologous End Joining ◽

Increasing Load

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The Maize Transposable Element Ac Induces Recombination Between the Donor Site and an Homologous Ectopic Sequence

Genetics ◽

10.1093/genetics/146.3.1143 ◽

1997 ◽

Vol 146 (3) ◽

pp. 1143-1151

Author(s):

Gil Shalev ◽

Avraham A Levy

Keyword(s):

Transposable Element ◽

Donor Site ◽

Double Strand Breaks ◽

Plant Genome ◽

Blue Staining ◽

Dna Double Strand Breaks ◽

End Joining ◽

Reading Frame ◽

Strand Breaks ◽

Ectopic Recombination

The prominent repair mechanism of DNA double-strand breaks formed upon excision of the maize Ac transposable element is via nonhomologous end joining. In this work we have studied the role of homologous recombination as an additional repair pathway. To this end, we developed an assay whereby β-Glucuronidase (GUS) activity is restored upon recombination between two homologous ectopic (nonallelic) sequences in transgenic tobacco plants. One of the recombination partners carried a deletion at the 5′ end of GUS and an Ac or a Ds element inserted at the deletion site. The other partner carried an intact 5′ end of the GUS open reading frame and had a deletion at the 3′ end of the gene. Based on GUS reactivation data, we found that the excision of Ac induced recombination between ectopic sequences by at least two orders of magnitude. Recombination events, visualized by blue staining, were detected in seedlings, in pollen and in protoplasts. DNA fragments corresponding to recombination events were recovered exclusively in crosses with Ac-carrying plants, providing physical evidence for Ac-induced ectopic recombination. The occurrence of ectopic recombination following double-strand breaks is a potentially important factor in plant genome evolution.

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Role for Artemis nuclease in the repair of radiation-induced DNA double strand breaks by alternative end joining

DNA Repair ◽

10.1016/j.dnarep.2015.04.004 ◽

2015 ◽

Vol 31 ◽

pp. 29-40 ◽

Cited By ~ 8

Author(s):

Mario Moscariello ◽

Radi Wieloch ◽

Aya Kurosawa ◽

Fanghua Li ◽

Noritaka Adachi ◽

...

Keyword(s):

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

End Joining ◽

Strand Breaks ◽

Alternative End Joining ◽

Radiation Induced

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Involvement of Ku80 in microhomology-mediated end joining for DNA double-strand breaks in vivo

DNA Repair ◽

10.1016/j.dnarep.2006.12.002 ◽

2007 ◽

Vol 6 (5) ◽

pp. 639-648 ◽

Cited By ~ 19

Author(s):

Yukitaka Katsura ◽

Shigeru Sasaki ◽

Masanori Sato ◽

Kiyoshi Yamaoka ◽

Kazumi Suzukawa ◽

...

Keyword(s):

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

End Joining ◽

Strand Breaks

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

The Journal of Cell Biology ◽

10.1083/jcb.200608077 ◽

2007 ◽

Vol 177 (2) ◽

pp. 219-229 ◽

Cited By ~ 256

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.

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