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 from Escherichia coli, human and herpes simplex virus have shown that two of the structural elements in Ugi, the hydrophobic pocket and the β1-edge, establish key interactions with UDGs. In this report the characterization of complexes of Ugi with UDGs from Mycobacterium tuberculosis, a pathogenic bacterium, and Mycobacterium smegmatis, a widely used model organism for the former, is described. Unlike the E. 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, while EcoUDG sequestered Ugi into the EcoUDG-Ugi complex when incubated with mycobacterial UDG-Ugi complexes, even a large excess of mycobacterial UDGs failed to sequester Ugi from the EcoUDG-Ugi complex. However, the M. tuberculosis (Mtu) UDG-Ugi complex was seen when MtuUDG was incubated with M. smegmatis (Msm) UDG-Ugi or EcoUDG(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 the β1-edge of Ugi) and with mutants of EcoUDG (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.

Synergistic effects of UdgB and Ung in mutation prevention and protection against commonly encountered DNA damaging agents in Mycobacterium smegmatis

“…As shown in Fig. 2(a), the purified protein (MSMEG_5031) excises uracil from GU9, a dsDNA substrate (S) containing uracil in a G : U pair at position 9 from the 59 end, and generates a product (P); and this activity is insensitive to the presence of Ugi, a highly specific Bacillus subtilis phage PBS1-encoded proteinaceous inhibitor (Acharya et al, 2003) of the family 1 UDGs (lanes 3-5). The MSMEG_5031-encoded protein did not show uracil excision activity on SSU9, an ssDNA substrate containing uracil within the same sequence context as dsDNA (lanes 1 and 2).…”

Yersiniophage ϕR1-37 is a tailed bacteriophage having a 270 kb DNA genome with thymidine replaced by deoxyuridine

“…This protein, uracil-DNA glycosylase inhibitor (Ugi), abolishes Ung activity in both bacteria and humans (Acharya et al, 2003;Olsen et al, 1991;Wang & Mosbaugh, 1988), and has been shown to increase mutation frequency when expressed in human cells (Radany et al, 2000). In addition to Ugi, PBS2 expresses a set of enzymes that function cooperatively to increase the intracellular dUTP pool and decrease the TTP concentration, thus resulting in the close to 100 % incorporation of dU instead of T in the DNA (Wang & Mosbaugh, 1988).…”

Virion-associated Uracil DNA Glycosylase-2 and Apurinic/Apyrimidinic Endonuclease Are Involved in the Degradation of APOBEC3G-edited Nascent HIV-1 DNA

“…If this hypothesis is correct, inhibition of virion-associated UNG2 could lead to less abasic site(s) in newly synthesized viral DNA chains and less degradation of newly synthesized viral DNA, and subsequently, less viral infectivity. Therefore we employed Ugi, a bacteriophage-encoded short polypeptide that can tightly bind to the DNA-binding site of UNG2, to inhibit the catalyzing activity of UNG2 in either virus-producing cells or viral target cells (23)(24)(25)(26)(27)(28). We have transfected Ugi-expressing vector in virus-producing 293T cells or viral target cells (HLCD4-CAT), respectively.…”