The Stl repressor from Staphylococcus aureus is an efficient inhibitor of the eukaryotic fruitfly dUTPase

Abstract

DNA metabolism and repair is vital for the maintenance of genome integrity. Specific proteinaceous inhibitors of key factors in this process have high potential for deciphering pathways of DNA metabolism and repair. The dUTP ase enzyme family is responsible for guarding against erroneous uracil incorporation into DNA . Here, we investigate whether the staphylococcal Stl repressor may interact with not only bacterial but also eukaryotic dUTP ase. We provide experimental evidence for the formation of a strong complex between Stl and Drosophila melanogaster dUTP ase. We also find that dUTP ase activity is strongly diminished in this complex. Our results suggest that the dUTP ase protein sequences involved in binding to Stl are at least partially conserved through evolution from bacteria to eukaryotes.

Structural model of human dUTPase in complex with a novel proteinaceous inhibitor

“…Remarkably, the catalytic activity of Φ11 dUTPase (Φ11DUT) is also inhibited upon complex formation with Stl, due to the competition of Stl and the substrate dUTP for dUTPase binding revealed by transient kinetic analysis 42 . Moreover, in our recent studies we have also demonstrated that Stl acts as a potent inhibitor of the Mycobacterial dUTPase, both in vitro and in vivo 21 and exerts inhibitory effect on the Drosophila melanoglaster dUTPase in vitro 43 .…”

“…aureus Φ 11 and 80 α phages, M . tuberculosis , and D. melanogaster in vitro 21 , 42 , 43 , although the latter two proteins share only modest sequence similarity with the original phage dUTPase partners of Stl. Using various techniques we have now demonstrated that Stl also interacts with the human dUTPase, which displays 62% sequence similarity to the phage dUTPases (Supplementary Fig.…”

“…smegmatis 21 insect Drosphila melanoglaster β-pleated trimer n.d. # max. 40% inhibition K i,app = 30 nM 43 n.d. # mammal human β-pleated trimer 230 nM (ITC) $ max. 70% inhibition $ K i,app = 6.7 nM $ n.d. # *Where available.…”

“…The 3:2 stoichiometry was also suggested for the complex of Φ11 phage and D . melanogaster dUTPases with Stl based on native MS 42 , 43 . It is important also to note that a higher order complex with Stl was suggested to exist for Φ11 phage dUTPase based on native gel electrophoresis results 42 .…”

Mass spectrometry‐based analysis of macromolecular complexes ofStaphylococcus aureusuracil‐DNA glycosylase and its inhibitor reveals specific variations due to naturally occurring mutations

“…Different strains of S. aureus encode numerous mutated versions of SAUGI [18]. While the exact biological role of SAUGI is still unclear, it is highly interesting to note that S. aureus also encodes an inhibitory protein for dUT-Pase, namely Stl [23][24][25][26][27].…”