TY - JOUR
T1 - Molecular basis of microhomology-mediated end-joining by purified full-length Polθ
AU - Black, Samuel J.
AU - Ozdemir, Ahmet Y.
AU - Kashkina, Ekaterina
AU - Kent, Tatiana
AU - Rusanov, Timur
AU - Ristic, Dejan
AU - Shin, Yeonoh
AU - Suma, Antonio
AU - Hoang, Trung
AU - Chandramouly, Gurushankar
AU - Siddique, Labiba A.
AU - Borisonnik, Nikita
AU - Sullivan-Reed, Katherine
AU - Mallon, Joseph S.
AU - Skorski, Tomasz
AU - Carnevale, Vincenzo
AU - Murakami, Katsuhiko S.
AU - Wyman, Claire
AU - Pomerantz, Richard T.
N1 - Funding Information:
This study was supported by the National Institutes of Health, grants to R.T.P. (1R01GM115472-01, 1R01GM130889-01), T.S. (1R01CA186238), and K.S.M. (R01GM087350). We are grateful to Drs. L. Prakash and S. Prakash for providing Polθ-pol purified from yeast. This research includes calculations carried out on Temple University’s HPC resources and thus was supported in part by the National Science Foundation through major research instrumentation grant number 1625061 and by the US Army Research Laboratory under contract number W911NF-16-2-0189.
Publisher Copyright:
© 2019, The Author(s).
PY - 2019/12/1
Y1 - 2019/12/1
N2 - 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.
AB - 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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U2 - 10.1038/s41467-019-12272-9
DO - 10.1038/s41467-019-12272-9
M3 - Article
C2 - 31562312
AN - SCOPUS:85072703007
SN - 2041-1723
VL - 10
JO - Nature Communications
JF - Nature Communications
IS - 1
M1 - 4423
ER -