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.
Highlights
polymerase θ (Polθ) consists of a super-family 2 helicase (Polθ-hel), a disordered central domain (Polθ-cen), and an A-family polymerase (Polθ-pol) (Fig. 1a and Supplementary Fig. 1)[1,10,11]
Initial biochemical studies demonstrated that short (≤ 15 nt) 3′ overhangs are required for MMEJ activity by the isolated polymerase domain (Polθ-pol)[2]
Consistent with this, we demonstrate that Polθ-pol exclusively performs single-strand DNA (ssDNA) extension when intrastrand base-pairing opportunities exist between the 3′ terminus and the 5′ portion of ssDNA (Supplementary Fig. 2)
Summary
Polθ consists of a super-family 2 helicase (Polθ-hel), a disordered central domain (Polθ-cen), and an A-family polymerase (Polθ-pol) (Fig. 1a and Supplementary Fig. 1)[1,10,11]. The polymerase and helicase include unstructured motifs, and a disordered motif (loop 2) in Polθ-pol was shown to promote its ssDNA extension and MMEJ activities[2,13,22]. Wyatt et al demonstrated that relatively long (≥ 45–70 nt) ssDNA overhangs promote MMEJ in cells (Fig. 1b). This is in agreement with prior reports showing that MMEJ and HR share the same 5′–3′ DNA resection pathway involving MRN and CtIP7, and that Polθ competes with HR3,15. This report further demonstrated that Ku binds tightly to DNA ends with short 4 nt overhangs, but exhibits very low affinity for 70 nt overhangs[8] This reveals a clear mechanistic difference between the respective substrate requirements for nonhomologous end-joining (NHEJ) and MMEJ. How Polθ functions on long ssDNA overhangs to promote MMEJ, remains unknown
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