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Black SJ, Ozdemir AY, Kashkina E, Kent T, Rusanov T, Ristic D, Shin Y, Suma A, Hoang T, Chandramouly G, Siddique LA, Borisonnik N, Sullivan-Reed K, Mallon JS, Skorski T, Carnevale V, Murakami KS, Wyman C, Pomerantz RT
Molecular basis of microhomology-mediated end-joining by purified full-length Poltheta
Nat Commun (2019) 10:4423.
Abstract
DNA polymerase theta (Poltheta) is a unique polymerase-helicase fusion protein that promotes microhomology-mediated end-joining (MMEJ) of DNA double-strand breaks (DSBs). How full-length human Poltheta performs MMEJ at the molecular level remains unknown. Using a biochemical approach, we find that the helicase is essential for Poltheta MMEJ of long ssDNA overhangs which model resected DSBs. Remarkably, Poltheta 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 Poltheta complexes that facilitate DNA accumulation, DNA synapsis, and MMEJ. We further find that the central domain regulates Poltheta 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 Poltheta.
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Publication Date: 2019-09-27.
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Last updated on Monday, November 04, 2019