The C-terminal region of translesion synthesis DNA polymerase η is partially unstructured and has high conformational flexibility

Abstract

Eukaryotic DNA polymerase η catalyzes translesion synthesis of thymine dimers and 8-oxoguanines. It is comprised of a polymerase domain and a C-terminal region, both of which are required for its biological function. The C-terminal region mediates interactions with proliferating cell nuclear antigen (PCNA) and other translesion synthesis proteins such as Rev1. This region contains a ubiquitin-binding/zinc-binding (UBZ) motif and a PCNA-interacting protein (PIP) motif. Currently little structural information is available for this region of polymerase η. Using a combination of approaches—including genetic complementation assays, X-ray crystallography, Langevin dynamics simulations, and small-angle X-ray scattering—we show that the C-terminal region is partially unstructured and has high conformational flexibility. This implies that the C-terminal region acts as a flexible tether linking the polymerase domain to PCNA thereby increasing its local concentration. Such tethering would facilitate the sampling of translesion synthesis polymerases to ensure that the most appropriate one is selected to bypass the lesion.

Cryo-EM structure of Pol κ−DNA−PCNA holoenzyme and implications for polymerase switching in DNA lesion bypass

“…The long region C-terminal to the internal PIP-box (residues 535-870) is invisible in the Cryo-EM map, suggesting it is largely disordered. In Pol h, flexibility of the C-terminal region has been previously observed experimentally 49 , and appears as a common characteristic of eukaryotic Yfamily polymerases 50 . The disordered C-terminal region of Pol k contains two UBZ zinc fingers (Figure 1a).…”

Modeling Conformationally Flexible Proteins With X-ray Scattering and Molecular Simulations

“…Nevertheless, this protein provides an excellent example to illustrate how predicted SAXS data are obtained from full ensembles. More detailed procedures for initial model building, molecular simulations, and data analysis are described elsewhere [ 51 ]. Rev1 has a complicated overall structure ( Fig.…”

“…Because of the large size of Rev1, all-atom MD simulations for several μs are not currently feasible. For this reason, the initial model was converted to a coarse-grained model as described previously [ 42 , 51 ]. Depending on the size, shape, and charge of the side chain, each amino acid residue was represented by one to four coarse-grain (CG) beads.…”

“…To model the conformational flexibility of the unstructured C-terminal region of pol η, coarse-grained LD simulations of full-length pol η were run for 10 μs of simulation time [ 51 ]. The predicted R g for the full ensemble was equal to 38.2 Å, and the predicted D max for the full ensemble was equal to 164 Å [ 51 ]. Overall, the C-terminal unstructured region sampled many conformational states, the vast majority of these were somewhere between largely extended states and largely compact ones.…”

“…Experimental SAXS data was used to validate the simulations, and excellent agreement was achieved between the LD simulations and the experimental SAXS data. For example, the experimental R g was equal to 37.6 Å, and the experimental D max was equal to 165 Å [ 51 ].…”