Fig 1: Molecular models of Polκ’s catalytic and noncatalytic functions during TLS.a,b, Colony formation assay with illudin S. U2OS parental cells, POLK-KO cells and POLK-KO cells stably expressing Venus–WT Polκ or Venus–CD Polκ were seeded at low density and cells expressing Venus–WT Polκ or Venus–CD Polκ were induced with 20 ng ml−1 doxycycline. Cells were grown in the presence or absence of 31.25 pg ml−1 illudin S and formed colonies were stained and quantified. Examples of images (a) and quantification (b) are shown. The survival in illudin S represents the average number of colonies at 31.25 pg ml−1 illudin S normalized to the untreated condition. Individual data points, means and s.d. values from three independent experiments are plotted. One-way ANOVA with Tukey’s multiple-comparisons test was performed and P values for the indicated comparisons are shown. c,d, Colony formation assay with cisplatin. Examples of images (c) and quantification (d) of a colony formation assay performed as in a,b but treated with 1 μM cisplatin. e,f, Composite molecular models of the catalytic (e) and noncatalytic (f) functions of Polκ during TLS. Models were generated by combining structure predictions from AlphaPulldown77 and AlphaFold2 (ref. 78), as described in the Methods. During its catalytic function (e), Polκ can interact with PCNA monomers through its PIP1 or PIP2 domains, bringing its catalytic domain into close proximity with the DNA substrate. The long flexible Polκ C-terminal region additionally allows Polκ to interact with a monoubiquitinated PCNA through its UBZ2 domain. During its noncatalytic function (f), Polκ can bind Rev1 through its RIR domain; the UBZ2 and PIP2 domains are able to bind to monoubiquitinated PCNA thanks to the long flexible Polκ C-terminal region. Notably, whilst the Polκ RIR domain is bound to Rev1, the PIP1 domain is positioned such that it would be unable to interact with monoubiquitylated PCNA. Note that the existence of a Rev1–Polζ–Polκ complex is inferred from a previous study47 and data presented in this manuscript.Source Data