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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 Sep 27;10(1) :4423
PMID: 31562312   
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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.
2041-1723 Black, Samuel J ORCID: http://orcid.org/0000-0001-5222-9024 Ozdemir, Ahmet Y Kashkina, Ekaterina Kent, Tatiana Rusanov, Timur Ristic, Dejan Shin, Yeonoh Suma, Antonio ORCID: http://orcid.org/0000-0002-5049-9255 Hoang, Trung Chandramouly, Gurushankar Siddique, Labiba A Borisonnik, Nikita Sullivan-Reed, Katherine Mallon, Joseph S Skorski, Tomasz ORCID: http://orcid.org/0000-0002-3809-4538 Carnevale, Vincenzo ORCID: http://orcid.org/0000-0002-1918-8280 Murakami, Katsuhiko S ORCID: http://orcid.org/0000-0003-2244-0501 Wyman, Claire Pomerantz, Richard T 1R01GM115472-01/U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS) Journal Article England Nat Commun. 2019 Sep 27;10(1):4423. doi: 10.1038/s41467-019-12272-9.