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 ? 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 UmuD2C/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 UmuD2C/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 UmuD2C/RecA model rationalizes published findings about UmuD residues in proximity to RecA. In summary, docking and molecular simulations are used to develop an UmuD2C/RecA model, whose structure rationalizes much of the known properties of the active form of DNA polymerase V.
b[a]p benzo[a]pyrene +bp [+ta]-b[a]p-n2-dg (fig. 1) ?bp [?ta]-b[a]p-n2-dg (fig. 1) tls translesion synthesis: the insertion of a base opposite a dna adduct as well as subsequent elongation dnap dna polymerase s1-dntp dntp active shape 1 s2-dntp dntp active shape 2 md molecular dynamics tt-cpd thyminethymine cyclopyrimidine dimer