Substrate specificities and functional characterization of a thermo-tolerant uracil DNA glycosylase (UdgB) from Mycobacterium tuberculosis.
Uracil DNA glycosylases (UDGs) excise uracil from DNA and initiate the base (uracil) excision repair pathway. Ung, a highly conserved protein, is the only UDG characterized so far in mycobacteria. Here, we show that Rv1259 from Mycobacterium tuberculosis codes for a double-stranded DNA (dsDNA) specific UDG (MtuUdgB). MtuUdgB is thermo-tolerant, contains Fe-S cluster and, in addition to uracil, it excises ethenocytosine and hypoxanthine from dsDNA. MtuUdgB is product inhibited by AP-site containing dsDNA but not by uracil. While MtuUdgB excises uracil present as a single-nucleotide bulge in dsDNA, it is insensitive to inhibition by dsDNA containing AP-site in the bulge. Interestingly, in the presence of cellular factors, the uracil excision activity of MtuUdgB is enhanced, and when introduced into E. coli (ung(-)), it rescues its mutator phenotype and prevents C to T mutations in DNA. Novel features of the mechanism of action of MtuUdgB and the physiological significance of the family 5 UDG in mycobacteria have been discussed.
Srinath
Thiruneelakantan
T
Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India.
Bharti
Sanjay Kumar
SK
Varshney
Umesh
U
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D024202:Electrophoretic Mobility Shift Assay; D004795:Enzyme Stability; D006358:Hot Temperature; D008969:Molecular Sequence Data; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D051981:Uracil-DNA Glycosidase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D017931:DNA Primers; D051981:Uracil-DNA Glycosidase