Abstract
Protein inhibitors of key DNA repair enzymes play an important role in deciphering physiological pathways responsible for genome integrity, and may also be exploited in biomedical research. The staphylococcal repressor StlSaPIbov1 protein was described to be an efficient inhibitor of dUTPase homologues showing a certain degree of species-specificity. In order to provide insight into the inhibition mechanism, in the present study we investigated the interaction of StlSaPIbov1 and Escherichia coli dUTPase. Although we observed a strong interaction of these proteins, unexpectedly the E. coli dUTPase was not inhibited. Seeking a structural explanation for this phenomenon, we identified a key amino acid position where specific mutations sensitized E. coli dUTPase to StlSaPIbov1 inhibition. We solved the three-dimensional (3D) crystal structure of such a mutant in complex with the substrate analogue dUPNPP and surprisingly found that the C-terminal arm of the enzyme, containing the P-loop-like motif was ordered in the structure. This segment was never localized before in any other E. coli dUTPase crystal structures. The 3D structure in agreement with solution phase experiments suggested that ordering of the flexible C-terminal segment upon substrate binding is a major factor in defining the sensitivity of E. coli dUTPase for StlSaPIbov1 inhibition.
Highlights
Preservation of genome integrity is of utmost importance for all living organisms
DNA synthesis before cell replication [19]. We recently discovered another potential route for perturbing dUTPase action via a protein inhibitor, StlSaPIbov1 [20]
We extended our Stl inhibition studies to E. coli dUTPase, since from this aspect it seems to be different from the Φ11 bacteriophage, human or mycobacterial enzymes for which Stl inhibition was observed [28,29,30]
Summary
Preservation of genome integrity is of utmost importance for all living organisms. Several complex pathways including numerous enzymes are involved in this duty. Absence of dUTPase (in differentiated tissues, knock-out models or several microorganisms) [4,5,6,7,8,9] or its inhibition combined with thymidylate synthase-targeted chemotherapies [10,11] is expected to lead to an increased level of uracil in DNA. Under such circumstances, DNA polymerases may readily incorporate uracil into DNA and uracil excision repair is activated.
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