Fig. 5
From: Molecular basis of microhomology-mediated end-joining by purified full-length Polθ
Cooperation of Polθ molecules and Polθ subdomains promotes ssDNA synapsis and MMEJ of long ssDNA. a Structural model of PolθΔcen on long ssDNA. PDB 4 × 0Q (Polθ-pol, blue), 5AGA (Polθ-hel, red). Surface (left) and ribbon (right) representations shown. b Exonuclease footprinting of PolθΔcen on ssDNA. Schematic of assay (left). Denaturing gel showing T5 exonuclease (0.1 U/ul) digestion of the indicated ssDNA with 10 nM Polθ-pol (lane 3), 10 nM PolθΔcen (lane 4), or no protein (lane 2). c EcoRI probing of PolθΔcen pssDNA binding. Schematic of assay (left). Non-denaturing gel showing EcoRI cleavage of the indicated pssDNA in the presence of the indicated proteins. d Schematic of FRET assay (left). Plot showing normalized FRET by the indicated proteins. n = 3 ± s.d. P values, unpaired Student’s two-sided t-test. e Dimeric model of PolθΔcen MMEJ of ssDNA with fluorophores. f Models of MMEJ involving Polθ monomers and dimers. g Polθ requires two long ssDNA substrates for MMEJ. Non-denaturing gel showing MMEJ by 3 nM of Polθ in the presence of the indicated ssDNA substrates (left). Table summarizing MMEJ data (right). Source data are provided as a Source Data file
