| Abstract |
Y-family DNA polymerases are important for conferring cellular resistance to DNA damaging agents in part due to their specialized ability to copy damaged DNA. The Escherichia coli Y-family DNA polymerases are encoded by the umuDC and dinB genes. UmuC and the cleaved form of UmuD, UmuD', form UmuD'2C (pol V), which is able to bypass UV photoproducts such as cyclobutane pyrimidine dimers and 6-4 thymine-thymine dimers, whereas DinB is specialized to copy N(2)-dG adducts, such as N(2)-furfuryl-dG. To better understand this inherent specificity, we used hydroxylamine to generate a random library of UmuC variants from which we then selected those with the ability to confer survival to nitrofurazone (NFZ), which is believed to cause N(2)-furfuryl-dG lesions. We tested the ability of three of the selected UmuC variants, A9V, H282P, and T412I, to bypass N(2)-furfuryl-dG in vitro, and discovered that pol V containing UmuC A9V has overall modestly better primer extension activity than WT pol V, whereas the UmuC T412I and UmuC H282P mutations result in much lower primer extension efficiency. Upon further characterization, we found that the ability of the UmuC variant A9V to render cells UV-mutable is dependent on the proper length of the arm of UmuD'. Cells harboring UmuC variants T412I and H282P show enhanced cleavage of UmuD to form UmuD', which, together with our other observations, suggests that this may be due to a disruption of a direct interaction between UmuC and UmuD. Thus, we find that protein interactions as well as protein conformation appear to be crucial for resistance to specific types of DNA damage.
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