Supplementary Figure 5: PrimPol realigns the primer-terminus ahead of the (6-4)pp lesion.
From: Repriming of DNA synthesis at stalled replication forks by human PrimPol
(a) TLS assay with a primer which hybridizes immediately before the (6-4)pp lesion (standing start). In an undamaged template, PrimPol elongated this primer until the expected +14 product (left panel; dNTPs: 1, 10 μM). In contrast, full length product was very scarce when copying the (6-4)pp-containing template (right panel). Instead, a main band was obtained corresponding to a +8 product, and a minor +11 band was also noticeable. (b) Standing start extension in the presence of the indicated dNTPs (10 μM), showing that dCTP is required for efficient primer extension in the (6-4)pp template. (c) Analysis of the first (+1) dNTP added to the primer in standing-start conditions. In the control template (left), dATP is the preferred nucleotide, whereas in the lesion-containing template (right), dCTP is preferentially inserted, with dA, dG and dT as secondary options. The bands corresponding to the primer (P) and elongation products (+1, +2, etc) are indicated. (d) Correlative insertion of dC, dT and dG, each provided at 1 mM, at control (left) and (6-4)pp-containing (right) templates. (e) Schematics summarizing the origin and relative abundance of the different products observed in (a) and (b). Direct extension allows the efficient copy of the normal template (+14); conversely, the small amount of the +14 product reflects inefficient synthesis opposite the (6-4)pp lesion. The (6-4)pp lesion (and the corresponding two Ts in the control template) is flanked by a 4 nt microhomology region (highlighted in yellow) that provides an opportunity to realign the primer-terminus as an alternative to copying the damaged bases (realignment beyond the lesion), explaining the most abundant +8 product on the (6-4)pp-containing template and the initial synthesis of CTG shown in (d). The same realignment could also take place in the control template, but is outcompeted by direct extension of the primer. Data are representative of 5 experiments (a), 3 experiments (b,c), 2 experiments (d).
