Uracil DNA glycosylase from Mycobacterium smegmatis and its distinct biochemical properties

Abstract

Deamination of cytosine residues contributes to the appearance of uracil in DNA. Uracil DNA glycosylase (UDG) initiates uracil excision repair to safeguard the genomic integrity. To study the mechanism of uracil excision in mycobacteria (organisms with GϩC rich genomes), we have purified UDG from Mycobacterium smegmatis by more than 3000-fold. The molecular mass of M. smegmatis UDG, as determined by SDS/PAGE, is Ϸ25 kDa and it shows maximum activity at pH 8.0. The N-terminal sequence analysis shows that the initiating amino acid, formyl-methionine is cleaved from the mature protein. More interestingly, unlike Escherichia coli UDG, which forms a physiologically irreversible complex with the inhibitor protein Ugi, M. smegmatis UDG forms a dissociable complex with it. M. smegmatis UDG excises uracil from the 5′-terminal position of the 5′-phosphorylated substrates. However, its excision from the 3′-penultimate position is extremely poor. Similar to E. coli UDG, M. smegmatis UDG also uses pd(UN)p as its minimal substrate. However, in contrast to E. coli UDG, which excises uracil from different loop positions of tetraloop hairpin substrates with highly variable efficiencies, M. smegmatis UDG excises the same uracil residues with comparable efficiencies. Kinetic parameters (K m and V max ) for uracil release from synthetic substrates suggest that M. smegmatis UDG is an efficient enzyme and better suited for molecular biology applications. We discuss the usefulness of the distinct biochemical properties of M. smegmatis UDG in the possible design of selective inhibitors against it.Keywords : UDG; Ung ; Ugi; hairpins; mycobacteria.Mycobacteria are responsible for a variety of public health problems. According to the recent WHO reports, a third of the world population is infected with Myobaceterium tuberculosis, and approximately 30 million deaths are likely to occur in the next 10 years on account of tuberculosis. Further, the emergence of drug-resistant strains has made it imperative to understand the biology of these organisms [1,2]. Mycobacteria multiply inside the host macrophages where they are exposed to oxidative or other physiological stresses [2Ϫ5]. Such adverse conditions result in damage to DNA [6,7]. However, the mechanisms of DNA repair in these important organisms have not been investigated so far. Mycobacteria are at increased risk of cytosine deamination not only because of their GϩC-rich genomes (up to 70%) but also because of the unfavorable habitat of the host macrophages where they multiply. Deamination of cytosine residues results in the appearance of uracil residues in DNA, which, unless repaired, lead to G :C→A:T mutations.Uracil DNA glycosylase (UDG) is responsible for uracil excision repair and crucial in safeguarding the genomic integrity [8Ϫ11]. UDGs characterized so far require no metal ions or other cofactors for their activity and can be divided into two groups. A number of diverse proteins such as the cyclin-like UDG [12] We are interested in the mechanism of uracil excision repair in mycob...

Importance of Uracil DNA Glycosylase in Pseudomonas aeruginosa and Mycobacterium smegmatis, G+C-rich Bacteria, in Mutation Prevention, Tolerance to Acidified Nitrite, and Endurance in Mouse Macrophages

“…Because a biochemical study (53) suggests weaker interaction between Ugi and M. smegmatis Ung (i.e. as compared with EcoUng), milder effects (Fig.…”

DNA repair inMycobacterium tuberculosisrevisited

“…Ung is important for the prevention of mutations, for increasing resistance to RNIs generated by acidified nitrite, and for the multiplication of GC‐rich bacteria within macrophages (in the M. smegmatis and Pseudomonas aeruginosa models) (Venkatesh et al , 2003). Like its E. coli counterpart, mycobacterial Ung efficiently excises uracil (Purnapatre & Varshney, 1998). Bacillus subtilis phage PBS‐1/2, which naturally contains uracil in its genome, encodes the early gene product, UDG inhibitor (Ugi).…”

A unique uracil-DNA binding protein of the uracil DNA glycosylase superfamily

“…The last UDG discovered belonged to the 6 th family, which represents hypoxanthine DNA glycosylase lacking uracil excision activity ( 9 ). Family 1 UDGs (Ung/UNG) are the most conserved and most extensively studied proteins of the UDG superfamily ( 8 , 14 – 17 ). Ung proteins, which are amongst the most efficient enzymes are characterized by motif A sequence, GQDPY involved in substrate catalysis, and motif B sequence, HPSPLS involved in stabilizing the enzyme substrate complex ( 3 , 18 – 20 ).…”