Structural model of the Y-Family DNA polymerase V/RecA mutasome

To synthesize past DNA damaged by chemicals or radiation, cells have lesion bypass DNA polymerases (DNAPs), most of which are in the Y-Family. One class of Y-Family DNAPs includes DNAP η in eukaryotes and DNAP V in bacteria, which have low fidelity when replicating undamaged DNA. In Escherchia coli, DNAP V is carefully regulated to insure it is active for lesion bypass only, and one mode of regulation involves interaction of the polymerase subunit (UmuC) and two regulatory subunits (UmuD’) with a RecA-filament bound to ss-DNA. Taking a docking approach, ∼150,000 unique orientations involving UmuC, UmuD’ and RecA were evaluated to generate models, one of which was judged best able to rationalize the following published findings. (1) In the UmuD’2C/RecA-filament model, R64-UmuC interacts with S117-RecA, which is known to be at the UmuC/RecA interface. (2) At the model's UmuC/RecA interface, UmuC has three basic amino acids (K59/R63/R64) that anchor it to RecA. No other Y-Family DNAP has three basic amino acids clustered in this region, making it a plausible site for UmuC to form its unique interaction with RecA. (3) In the model, residues N32/N33/D34 of UmuC form a second interface with RecA, which is consistent with published findings. (4) Active UmuD’ is generated when 24 amino acids in the N-terminal tail of UmuD are proteolyzed, which occurs when UmuD2C binds the RecA-filament. When UmuD is included in an UmuD2C/RecA-filament model, plausible UmuD/RecA contacts guide the UmuD cleavage site (C24/G25) into the UmuD proteolysis active site (S60/K97). One contact involves E11-UmuD interacting with R243-RecA, where the latter is known to be important for UmuD cleavage. (5) The UmuD2C/RecA-filament model rationalizes published findings that at least some UmuD-to-UmuD’ cleavage occurs intermolecularly. (6) Active DNAP V is known to be the heterotetramer UmuD’2C/RecA, a model of which can be generated by a simple rearrangement of the RecA monomer at the 3′-end of the RecA-filament. The rearranged UmuD’2C/RecA model rationalizes published findings about UmuD’ residues in proximity to RecA. In summary, docking and molecular simulations are used to develop an UmuD’2C/RecA model, whose structure rationalizes much of the known properties of the active form of DNA polymerase V.

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► Y-Family DNA polymerase V models explain most experimental findings.
► Model for DNA polymerase V at a RecA filament prior to UmuD cleavage.
► Structural model for UmuD cleavage to give active DNA polymerase V.
► Model for active DNA polymerase V, which is the tetramer UmuD’2C/RecA.