title: The oxidative DNA glycosylases of Mycobacterium tuberculosis exhibit different substrate preferences from their Escherichia coli counterparts.
abstract: The DNA glycosylases that remove oxidized DNA bases fall into two general families: the Fpg/Nei family and the Nth superfamily. Based on protein sequence alignments, we identified four putative Fpg/Nei family members, as well as a putative Nth protein in Mycobacterium tuberculosis H37Rv. All four Fpg/Nei proteins were successfully overexpressed using a bicistronic vector created in our laboratory. The MtuNth protein was also overexpressed in soluble form. The substrate specificities of the purified enzymes were characterized in vitro with oligodeoxynucleotide substrates containing single lesions. Some were further characterized by gas chromatography/mass spectrometry (GC/MS) analysis of products released from gamma-irradiated DNA. MtuFpg1 has substrate specificity similar to that of EcoFpg. Both EcoFpg and MtuFpg1 are more efficient at removing spiroiminodihydantoin (Sp) than 7,8-dihydro-8-oxoguanine (8-oxoG). However, MtuFpg1 shows a substantially increased opposite base discrimination compared to EcoFpg. MtuFpg2 contains only the C-terminal domain of an Fpg protein and has no detectable DNA binding activity or DNA glycosylase/lyase activity and thus appears to be a pseudogene. MtuNei1 recognizes oxidized pyrimidines on both double-stranded and single-stranded DNA and exhibits uracil DNA glycosylase activity. MtuNth recognizes a variety of oxidized bases, including urea, 5,6-dihydrouracil (DHU), 5-hydroxyuracil (5-OHU), 5-hydroxycytosine (5-OHC) and methylhydantoin (MeHyd). Both MtuNei1 and MtuNth excise thymine glycol (Tg); however, MtuNei1 strongly prefers the (5R) isomers, whereas MtuNth recognizes only the (5S) isomers. MtuNei2 did not demonstrate activity in vitro as a recombinant protein, but like MtuNei1 when expressed in Escherichia coli, it decreased the spontaneous mutation frequency of both the fpg mutY nei triple and nei nth double mutants, suggesting that MtuNei2 is functionally active in vivo recognizing both guanine and cytosine oxidation products. The kinetic parameters of the MtuFpg1, MtuNei1 and MtuNth proteins on selected substrates were also determined and compared to those of their E. coli homologs.
authors.affiliation: Department of Microbiology and Molecular Genetics, The Markey Center for Molecular Genetics, University of Vermont, Stafford Hall, 95 Carrigan Dr., Burlington, VT 05405-0068, United States.
authors.forename: Yin
authors.initials: Y
authors.lastname: Guo
authors.forename: Viswanath
authors.initials: V
authors.lastname: Bandaru
authors.forename: Pawel
authors.initials: P
authors.lastname: Jaruga
authors.forename: Xiaobei
authors.initials: X
authors.lastname: Zhao
authors.forename: Cynthia J
authors.initials: CJ
authors.lastname: Burrows
authors.forename: Shigenori
authors.initials: S
authors.lastname: Iwai
authors.forename: Miral
authors.initials: M
authors.lastname: Dizdaroglu
authors.forename: Jeffrey P
authors.initials: JP
authors.lastname: Bond
authors.forename: Susan S
authors.initials: SS
authors.lastname: Wallace
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism*; D045647:DNA Glycosylases / Q000378:metabolism*; D004269:DNA, Bacterial / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism; D024881:Drug Resistance, Bacterial / Q000187:drug effects; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology*; D008401:Gas Chromatography-Mass Spectrometry; D007700:Kinetics; D009154:Mutation / Q000235:genetics; D009169:Mycobacterium tuberculosis / Q000187:drug effects / Q000201:enzymology*; D010084:Oxidation-Reduction / Q000187:drug effects; D011544:Pseudogenes / Q000235:genetics; D011687:Purines / Q000378:metabolism; D011743:Pyrimidines / Q000378:metabolism; D012293:Rifampin / Q000494:pharmacology; D013237:Stereoisomerism; D013379:Substrate Specificity / Q000187:drug effects
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D011687:Purines; D011743:Pyrimidines; D045647:DNA Glycosylases; D012293:Rifampin