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'(2)C/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 UmuD(2)C binds the RecA-filament. When UmuD is included in an UmuD(2)C/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 UmuD(2)C/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'(2)C/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'(2)C/RecA model rationalizes published findings about UmuD' residues in proximity to RecA. In summary, docking and molecular simulations are used to develop an UmuD'(2)C/RecA model, whose structure rationalizes much of the known properties of the active form of DNA polymerase V.
[Show abstract][Hide abstract] ABSTRACT: The presence of unrepaired lesions in DNA represents a challenge for replication. Most, but not all, DNA lesions block the replicative DNA polymerases. The conceptually simplest procedure to bypass lesions during DNA replication is translesion synthesis (TLS), whereby the replicative polymerase is transiently replaced by a specialized DNA polymerase that synthesizes a short patch of DNA across the site of damage. This process is inherently error prone and is the main source of point mutations. The diversity of existing DNA lesions and the biochemical properties of Escherichia coli DNA polymerases will be presented. Our main goal is to deliver an integrated view of TLS pathways involving the multiple switches between replicative and specialized DNA polymerases and their interaction with key accessory factors. Finally, a brief glance at how other bacteria deal with TLS and mutagenesis is presented.
Cold Spring Harbor perspectives in biology 12/2013; 5(12). DOI:10.1101/cshperspect.a012682 · 8.68 Impact Factor
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