Fig. 5: Mutational analysis of the Pol κ synthetic activity.

a Schematic view of the Pol κ mutants used in the primer extension assays. Mutated residues are colored red above the native sequence. b Schematic representation of the primer/template DNA substrate used in the assays. c, d Activity of Pol κ and stimulation by PCNA on the DNA substrate. Wild-type Pol κ and Pol κ mutants were incubated with the substrate in the presence and absence of PCNA at 30 °C for 40 s and the products were separated on 15% denaturing polyacrylamide gels at 12 W for 3 h. The reaction’s procedure is detailed in the “Methods” section. Experiments have been repeated at least two times. e Plots of the cumulative percentages of DNA synthesis up to the indicated number of synthesized nucleotides constructed from the lanes of the gels presented in panels (c) and (d), respectively. The curves were generated as described in the “Methods” section. The experimental data points (circles) were fitted to relatively rough smoothing splines (solid lines) with the indicated smoothing parameter. The curves account exclusively for the distribution of the bands corresponding to DNA synthesis products and not for the remaining enzymatically unmodified substrate. In both graphs, the horizontal black dashed lines represent a median DNA synthesis value of 50%. The intersection of each smoothing spline fit with this 50% line gives an N_1/2 value, which represents an apparent number of synthesized nucleotides up to which 50% of the total DNA synthesis is achieved. For both graphs, the experimental data points and their corresponding fitting curves are color-coded as presented in the inset legend. In both graphs, the black star sign marks the values corresponding to 27 synthesized DNA nt to highlight that this value was not obtained directly but rather represents a summation of the smeared region between 21 and 33 nt following background subtraction. f (top) Alignment of the sequences of human Rad18-UBZ domains and Pol κ UBZ domains from mouse and human. Zinc-coordinating residues are labeled with Δ. Residues at the interface with ubiquitin are labeled with asterisks. Conserved residues by type are colored gray. (bottom) Homology model of human Pol κ UBZ1 aligned on the NMR structure of Rad18-UBZ/Ubiquitin complex (PDB ID 2MRE)⁵¹. Pol κ UBZ1 model was built with HHpred⁹⁵ based on the Rad18-UBZ/ubiquitin complex structure⁵¹. Residues involved in the salt bridge critical for UBZ/Ubiquitin complex formation are shown as sticks.