Downloaded from www.microbiologyresearch.org by IP: 52.90.13.16 On: Sun, 05 Nov 2017 14:10:18 Complexes of the uracil-DNA glycosylase inhibitor protein, Ugi, with Mycobacterium smegmatis and Mycobacterium tuberculosis uracil-DNA glycosylases Narottam Acharya, Pradeep Kumar and Umesh Varshney Correspondence Umesh Varshney varshney@mcbl.iisc.ernet.in Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, 560 012, India Received 9 January 2003 Revised 26 March 2003 Accepted 26 March 2003 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 b1-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 b1-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. INTRODUCTION The specific recognition and removal of an RNA base, uracil, in DNA is the province of a highly specialized DNA repair enzyme, uracil-DNA glycosylase (UDG) (Lindahl, 1974; Krokan et al., 1997). These enzymes belong to a highly conserved ubiquitous class of proteins (Aravind & Koonin, 2000). Interestingly, Bacillus subtilis phage PBS-1/2 which naturally contains uracil in its genome, encodes an early gene product, UDG inhibitor (Ugi) to protect its genome from host UDG by forming an extremely specific and exclusively stable complex. Ugi is a small (84 aa, 9?4 kDa), highly acidic (pI 4?2) and thermostable protein which interacts with UDGs in 1 : 1 molar stoichiometry (Cone et al., 1980; Wang & Mosbaugh, 1989; Savva & Pearl, 1995; Mol et al., 1995). Biochemical characterization of the Escherichia coli (Eco) UDG-Ugi complex has shown it to be irreversible under physiological conditions (Bennett & Mosbaugh, 1992). Furthermore, a stopped flow kinetic study suggested a two-step (‘docking’ and ‘locking’) mechanism of EcoUDG-Ugi complex formation (Bennett et al., 1993). The co-crystal structures of various UDGs with Ugi have been resolved and show an extraordinary con- servation in their overall architecture (Savva & Pearl, 1995; Mol et al., 1995; Ravishankar et al., 1998; Putnam et al., 1999). Interestingly, these studies have identified Ugi as a transition-state substrate mimic, making it an attractive model system to understand the mechanistic aspects of protein–protein interaction. Ugi primarily uses two of its structural elements, the hydrophobic pocket between the a2 helix and the antiparallel b sheet, and the unusually shaped Abbreviations: HSV, herpes simplex virus; UDG, uracil-DNA glycosy- lase; Ugi, uracil-DNA glycosylase inhibitor. 0002-6228 G 2003 SGM Printed in Great Britain 1647 Microbiology (2003), 149, 1647–1658 DOI 10.1099/mic.0.26228-0