UVM Theses and Dissertations
Format:
Online
Author:
Guo, Yin
Dept./Program:
Microbiology and Molecular Genetics
Year:
2010
Degree:
PhD
Abstract:
The DNA glycosylases function in the first step ,of the base excision repair (BER) process, that is responsible for removing base lesions resulting from oxidation, alkylation or deamination. The DNA glycosylases that recognize oxidative base damage fall into two general families: the FpglNei 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 y-proteobacteria, actinobacteriaand metazoans. Interestingly, M tuberculosis H37Rv harbors two proteins (Rv2464c and, Rv3297) from the Nei clade and two (Rv2924canq Rv0944) from the Fpg clade.
All four Fpg/Nei proteins were successfully overexpressed by using a novel bicistronic vector, which theoretically prevented stable mRNA secondary structure(s) surrounding the translation initiation region (TIR) thereby improving translation efficiency. Additionally, MtuNth (Rv3674c) was also overexpressed in soluble form. The substrate specificities of the purified enzymes were characterized in vitro with oligonucleotide substrates containing single lesions. Some were further characterized by gas chromatography/mass spectrometry (GCIMS) analysis of products released from y-irradiated DNA. MtuFpgl (Rv2924c) has a substrate specificity similar to that of Eco;Fpg and recognizes oxidized purines. Both EcoFpg and MtuFpgI are more effident at'removing spiroiminodihydantoin (Sp) than 7,8-dihydro-8-oxoguanine (8-oxoG); however, MtuFpgl has a substantially increased opposite base, discrimination compared to EcoFpg.
The Rv0944 gene encodes MtuFpg2, which, 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. MtuNeil (Rv2464c) recognizes oxidized pyrimidines not only on doublestranded DNA but also on single-stranded DNA. It also exhibits uracil DNA glycosylase/lyase activity as well as weak activity on FapyA and FapyG. MtuNth recognizes a variety of oxidized bases, such as urea, 5,6-dihydrouracil (DHU), 5-hydroxyuracil (5-OHU), MtuNth recognizes a variety of oxidized bases, such as urea, 5,6-dihydrouracil (DHU), 5-hydroxyuracil (5OHU), 5-hydroxycytosine (5-OHC) and methylhydantoin (MeHyd) as well as FapyA, FapyG and 8-oxoadenine (8-oxoA). Both MtuNeil and MtuNth excise thymine glycol (Tg); however, MtuNeil strongly prefers the (5R) isomers of Tg, whereas MtuNth recognizes only the (58) isomers. The other Nei paralog, MtuNei2 (Rv3297), did not demonstrate activity in vitro as a recombinant protein, but when expressed in Esche richia coli, the protein 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, MtuNeil and MtuNth proteins on selected substrates were also determined and compared to those of their E. coli homologs. Since pathogenic bacteria are often exposed to an oxidative environment, such as in macrophages, our data, together with previous observations, support the idea that the BER pathway is of importance in protecting M tuberculosis against oxidative stress, as has been observed with other pathogens.
All four Fpg/Nei proteins were successfully overexpressed by using a novel bicistronic vector, which theoretically prevented stable mRNA secondary structure(s) surrounding the translation initiation region (TIR) thereby improving translation efficiency. Additionally, MtuNth (Rv3674c) was also overexpressed in soluble form. The substrate specificities of the purified enzymes were characterized in vitro with oligonucleotide substrates containing single lesions. Some were further characterized by gas chromatography/mass spectrometry (GCIMS) analysis of products released from y-irradiated DNA. MtuFpgl (Rv2924c) has a substrate specificity similar to that of Eco;Fpg and recognizes oxidized purines. Both EcoFpg and MtuFpgI are more effident at'removing spiroiminodihydantoin (Sp) than 7,8-dihydro-8-oxoguanine (8-oxoG); however, MtuFpgl has a substantially increased opposite base, discrimination compared to EcoFpg.
The Rv0944 gene encodes MtuFpg2, which, 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. MtuNeil (Rv2464c) recognizes oxidized pyrimidines not only on doublestranded DNA but also on single-stranded DNA. It also exhibits uracil DNA glycosylase/lyase activity as well as weak activity on FapyA and FapyG. MtuNth recognizes a variety of oxidized bases, such as urea, 5,6-dihydrouracil (DHU), 5-hydroxyuracil (5-OHU), MtuNth recognizes a variety of oxidized bases, such as urea, 5,6-dihydrouracil (DHU), 5-hydroxyuracil (5OHU), 5-hydroxycytosine (5-OHC) and methylhydantoin (MeHyd) as well as FapyA, FapyG and 8-oxoadenine (8-oxoA). Both MtuNeil and MtuNth excise thymine glycol (Tg); however, MtuNeil strongly prefers the (5R) isomers of Tg, whereas MtuNth recognizes only the (58) isomers. The other Nei paralog, MtuNei2 (Rv3297), did not demonstrate activity in vitro as a recombinant protein, but when expressed in Esche richia coli, the protein 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, MtuNeil and MtuNth proteins on selected substrates were also determined and compared to those of their E. coli homologs. Since pathogenic bacteria are often exposed to an oxidative environment, such as in macrophages, our data, together with previous observations, support the idea that the BER pathway is of importance in protecting M tuberculosis against oxidative stress, as has been observed with other pathogens.