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

Samuel J. Black, Ahmet Y. Ozdemir, Ekaterina Kashkina, Tatiana Kent, Timur Rusanov, Dejan Ristic, Yeonoh Shin, Antonio Suma, Trung Hoang, Gurushankar Chandramouly, Labiba A. Siddique, Nikita Borisonnik, Katherine Sullivan-Reed, Joseph S. Mallon, Tomasz Skorski, Vincenzo Carnevale, Katsuhiko S. Murakami, Claire Wyman and Richard T. Pomerantz ()
Additional contact information
Samuel J. Black: Temple University Lewis Katz School of Medicine
Ahmet Y. Ozdemir: Temple University Lewis Katz School of Medicine
Ekaterina Kashkina: Temple University Lewis Katz School of Medicine
Tatiana Kent: Temple University Lewis Katz School of Medicine
Timur Rusanov: Temple University Lewis Katz School of Medicine
Dejan Ristic: Erasmus University Medical Center
Yeonoh Shin: Pennsylvania State University
Antonio Suma: Institute for Computational Molecular Science, Temple University
Trung Hoang: Temple University Lewis Katz School of Medicine
Gurushankar Chandramouly: Temple University Lewis Katz School of Medicine
Labiba A. Siddique: Temple University Lewis Katz School of Medicine
Nikita Borisonnik: Temple University Lewis Katz School of Medicine
Katherine Sullivan-Reed: Temple University Lewis Katz School of Medicine
Joseph S. Mallon: Temple University Lewis Katz School of Medicine
Tomasz Skorski: Temple University Lewis Katz School of Medicine
Vincenzo Carnevale: Institute for Computational Molecular Science, Temple University
Katsuhiko S. Murakami: Pennsylvania State University
Claire Wyman: Erasmus University Medical Center
Richard T. Pomerantz: Temple University Lewis Katz School of Medicine

Nature Communications, 2019, vol. 10, issue 1, 1-16

Abstract: 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.

Date: 2019
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DOI: 10.1038/s41467-019-12272-9

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