Complexes of the uracil-DNA glycosylase inhibitor protein, Ugi, with Mycobacterium smegmatis and Mycobacterium tuberculosis uracil-DNA glycosylases

Abstract

Uracil, a promutagenic base, appears in DNA either by deamination of cytosine or by incorporation of dUMP by DNA polymerases. This unconventional base in DNA is removed by uracil-DNA glycosylase (UDG). Interestingly, a bacteriophage-encoded short polypeptide, UDG inhibitor (Ugi), specifically inhibits UDGs by forming a tight complex. Three-dimensional structures of the complexes of Ugi with UDGs fromEscherichia coli, human and herpes simplex virus have shown that two of the structural elements in Ugi, the hydrophobic pocket and theβ1-edge, establish key interactions with UDGs. In this report the characterization of complexes of Ugi with UDGs fromMycobacterium tuberculosis, a pathogenic bacterium, andMycobacterium smegmatis, a widely used model organism for the former, is described. Unlike theE. coli(Eco) UDG-Ugi complex, which is stable to treatment with 8 M urea, the mycobacterial UDG-Ugi complexes dissociate in 5–6 M urea. Furthermore, the Ugi from the complexes of mycobacterial UDGs can be exchanged by the DNA substrate. Interestingly, whileEcoUDG sequestered Ugi into theEcoUDG-Ugi complex when incubated with mycobacterial UDG-Ugi complexes, even a large excess of mycobacterial UDGs failed to sequester Ugi from theEcoUDG-Ugi complex. However, theM. tuberculosis(Mtu) UDG-Ugi complex was seen whenMtuUDG was incubated withM. smegmatis(Msm) UDG-Ugi orEcoUDG(L191G)-Ugi complexes. The reversible nature of the complexes of Ugi with mycobacterial UDGs (which naturally lack some of the structural elements important for interaction with theβ1-edge of Ugi) and with mutants ofEcoUDG (which are deficient in interaction with the hydrophobic pocket of Ugi) highlights the significance of both classes of interaction in formation of UDG-Ugi complexes. Furthermore, it is shown that even though mycobacterial UDG-Ugi complexes dissociate in 5–6 M urea, Ugi is still a potent inhibitor of UDG activity in mycobacteria.