Fig. 4: Second-strand repair by NHEJ.
From: Nonhomologous end-joining uses distinct mechanisms to repair each strand of a double strand break
a Substrate employed to focus on repair of gap in 2nd strand of substrate in Fig. 3. b The substrate on right from panel awas introduced into MEFs deficient in Pol μ only (Polm-/-), or in all three members of the Pol X family (PolX-/-), or PolX-/- deficient cells complemented by introduction of equal amounts of Pol β (1xWT β, 1x = 250 ng), Pol μ (1xWT μ), Pol λ (1xWT λ), Pol λ L60A (1xL60A λ), or 3-fold more Pol λ L60A (3xL60A λ). A vertical dashed line separates experiments that were performed on different days. DNA was harvested after 5 min in cells. The abundance of top strand products with the noted +C product, as well as the abundance of bottom strands with the complementary sequence, was determined by digital PCR. The fraction of both strands joined was expressed as the ratio of bottom strands with complementary sequence over top strand +C products, ±s.d. from n = 3 independent biological replicates, with the exception of PolX-/- +1x WT Pol λ (left of dashed line), including two replicates. Means were compared by one-way ANOVA with Dunnett’s test for multiple comparisons for each experiment independently (each side of the dashed line). ****p < 0.0001, ns = not significant. Source data for all relevant panels are provided as a Source Data file. c 1nt-gapped SSB DNA intermediate substrate with template strand embedded ribonucleotide (magenta) co-crystallized with the Pol λ catalytic domain and an incoming nonhydrolyzable dUMPNPP nucleotide (cyan). d View of the pre-catalytic SSB active site (protein in blue, template DNA strand in khaki, upstream primer strand in green, incoming nucleotide in cyan) with secondary structures depicted as ribbons, and bound substrates in stick. The Na+ ion bound in the HhH2, and the active site divalent Mg2+ ions are shown as yellow or green spheres, respectively. 2Fo-Fc electron density is drawn in gray mesh (contoured at 1σ). e Superposition of the 2nd-strand gap-filling intermediate (khaki) containing template-embedded ribonucleotide (magenta) with the same substate but containing a deoxyribonucleotide at the same position (gray, PDB ID code: 7M07 [https://doi.org/10.1038/s41467-022-31278-4])26, highlighting slight structural variations in the template strand due to the presence of the ribonucleotide. Positional shifts and alterations in sugar puckering are marked by black arrows. f Zoomed-in view of the upstream primer terminal (light green) and incoming dUMPNPP (cyan) nucleotides, highlighting the mixture of conformations for the 3ʹ-OH.
