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 γ-irradiated DNA. MtuFpg1 has a 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 b Current address: Mylan Pharmaceuticals, 3711 Collins Ferry Rd, Morgantown, WV 26505Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access

Two glycosylase families diffusively scan DNA using a wedge residue to probe for and identify oxidatively damaged bases

“…Although the catalytic activity of Fpg on Tg damage may be low, the enzyme can still recognize Tg; thus, the binding lifetime on OsO 4 -treated λ-DNA is prolonged (2.0 ± 0.4 s) (P < 0.001) compared with undamaged DNA (1.2 ± 0.1 s) but not to the same extent as on MB-treated DNA (3.5 ± 0.9 s) (Fig. 5B), which is in keeping with the ensemble studies (39).…”

“…5). The oxidative damage produced by OsO 4 treatment (i.e., Tg opposite A) is a poor substrate for Fpg as its catalytic efficiency on this oxidized pyrimidine is 20-fold less than its activity on 8-oxoG and over 100-fold less that of Nei on Tg (39). As expected, the mean overall diffusion constant, D traj , for WT Fpg on OsO 4 -treated λ-DNA (0.002 ± 0.006 μm 2 /s) was the same as that observed on undamaged DNA (0.003 ± 0.001 μm 2 /s) (P > 0.4), whereas diffusive motion of Fpg on MB-treated DNA was significantly lower (0.00065 ± 0.00022 μm 2 /s) (P < 0.001) (Fig.…”

The mouse ortholog of NEIL3 is a functional DNA glycosylase in vitro and in vivo

“…is comparable to that of EcoFpg (0.24-5.05 min −1 nM −1 (22,24)), and higher than EcoNei (0.05-1.39 min −1 nM −1 (22,24)). Like NEIL1 and NEIL2, MmuNeil3Δ324 prefers singlestranded DNA or partially single-stranded DNA structures such as bubble and fork structures (Fig.…”

“…S4) suggested that although MmuNeil3Δ324 is bifunctional (with rates of 0.119AE 0.016 min −1 for single-stranded Sp1 and 0.053 AE 0.002 min −1 for double-stranded Sp1 substrates), its lyase activity is not coupled to its glycosylase activity, since the slow lyase reaction of MmuNeil3Δ324 does not limit the enzyme turnover of the glycosylase reaction. MmuNeil3Δ324 has a similar catalytic turnover rate for the glycosylase reaction (3.2 min −1 , Table 1) on double-stranded Sp substrate as does E. coli Nei (1.6-13.0 min −1 (22)(23)(24)) and Fpg (6.0-34.2 min −1 (22)(23)(24)) proteins, although it is much slower than NEIL1 (177 min −1 (25)) on the same substrate. However, MmuNeil3Δ324 exhibits a much higher binding affinity to a double-stranded Sp substrate (K m ¼ 0.753 nM, Table 1), compared to EcoFpg (K m ¼ 25 nM (24)) and EcoNei (K m ¼ 30 nM (24)).…”

Biochemical Properties of MutT2 Proteins from Mycobacterium tuberculosis and M. smegmatis and Their Contrasting Antimutator Roles in Escherichia coli

“…Fpg excises 8-oxo-G from DNA and initiates base excision repair (BER) to replace the damaged base with a normal base (12,13). If a lapse in the removal of 8-oxo-G leads to incorporation of A against 8-oxo-G, another BER enzyme, MutY, removes the A from the 8-oxo-G-A mispairs to incorporate C against 8-oxo-G to allow Fpg to work again (14).…”