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The oxidative DNA glycosylases of Mycobacterium tuberculosis exhibit different substrate specificities from their Escherichia coli counterparts

Published

Author(s)

Yin Guo, Viswanath Bandaru, Pawel Jaruga, Xiaobei Zhao, Cynthia Burrows, Shigenori Iwai, Miral M. Dizdar, Jeffrey Bond, Susan Wallace

Abstract

The DNA glycosylases function in the first step of the base excision repair process that is responsible for removing endogenous oxidative purine and pyrimidine damages from DNA. 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, the causative agent of tuberculosis. While Fpg proteins are widely distributed among the bacteria and plants, Nei homologs are sparsely distributed across phyla, and are only found in -proteobacteria, actinobacteria and metazoans. Interestingly, in M. tuberculosis H37Rv, two proteins were identified from the Nei clade and two from the Fpg clade. All four proteins were successfully overexpressed by 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 -irradiated DNA. MtuFpg1 has a substrate specificity similar to that of EcoFpg and recognizes oxidized purines, including: 7,8-dihydro-8-oxoguanine (8-oxoG), 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG), and 4,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyA), although the latter less so than EcoFpg. MtuFpg1 has 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 not only on double-stranded but also on single-stranded DNA and exhibits uracil DNA glycosylase activity as well as weak activity on FapyA and Fap
Citation
Dna Repair
Volume
9
Issue
2

Keywords

Base Excision Repair, DNA Glycosylases, Formamidopyrimidine DNA Glycosylase, Mycobacterium tuberculosis

Citation

Guo, Y. , Bandaru, V. , Jaruga, P. , Zhao, X. , Burrows, C. , Iwai, S. , Dizdar, M. , Bond, J. and Wallace, S. (2010), The oxidative DNA glycosylases of Mycobacterium tuberculosis exhibit different substrate specificities from their Escherichia coli counterparts, Dna Repair (Accessed March 4, 2024)
Created February 3, 2010, Updated October 12, 2021