Substrate specificities and functional characterization of a thermo-tolerant uracil DNA glycosylase (UdgB) from Mycobacterium tuberculosis

DNA Repair (Amst). 2007 Oct 1;6(10):1517-28. doi: 10.1016/j.dnarep.2007.05.001. Epub 2007 Jun 27.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Base Sequence
  • Cloning, Molecular
  • DNA Primers
  • Electrophoretic Mobility Shift Assay
  • Enzyme Stability
  • Hot Temperature
  • Molecular Sequence Data
  • Mutation
  • Mycobacterium tuberculosis / enzymology*
  • Sequence Homology, Amino Acid
  • Substrate Specificity
  • Uracil-DNA Glycosidase / chemistry
  • Uracil-DNA Glycosidase / genetics
  • Uracil-DNA Glycosidase / isolation & purification
  • Uracil-DNA Glycosidase / metabolism*

Substances

  • DNA Primers
  • Uracil-DNA Glycosidase