Repair of oxidized bases in the extremely radiation-resistant bacterium Deinococcus radiodurans - PubMed (original) (raw)

Repair of oxidized bases in the extremely radiation-resistant bacterium Deinococcus radiodurans

C Bauche et al. J Bacteriol. 1999 Jan.

Abstract

Deinococcus radiodurans is able to resist and survive extreme DNA damage induced by ionizing radiation and many other DNA-damaging agents. It is believed that it possesses highly efficient DNA repair mechanisms. To characterize the repair pathway of oxidized purines in this bacteria, we have purified, from crude extracts, proteins that recognize these oxidized bases. We report here that D. radiodurans possesses two proteins excising the oxidized purines (formamidopyrimidine and 8-oxoguanine) by a DNA glycosylase-a purinic/apyrimidine lyase mechanism. Moreover, one of those proteins is endowed with a thymine glycol DNA glycosylase activity. One of these proteins could be the homolog of the Escherichia coli Fpg enzyme, which confirms the existence of a base excision repair system in this bacteria.

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Figures

FIG. 1

FIG. 1

Cleavage of the 8-oxoG/C duplex by the following: lane 1, no enzyme, lane 2, 5 ng of E. coli Fpg protein; lanes 3, 4, and 5, 0.2, 0.4, and 1 U of Fapy DNA glycosylase (fraction V); lanes 6, 7, and 8, 0.2, 0.4, and 1 U of Fapy DNA glycosylase (fraction IV). The duplex (100 fmol/reaction) was incubated with the enzyme at 32°C for 10 min.

FIG. 2

FIG. 2

Immunoblotting of fractions V and VI protein with anti-Fpg (A) and anti-Nth (B) antibodies. Lane 1, E. coli Fpg protein (100 ng); lane 2, E. coli Nth protein (100 ng); lane 3, fraction V (0.5 U of Fapy-DNA glycosylase); lane 4, fraction VI (0.5 U of Fapy-DNA glycosylase).

FIG. 3

FIG. 3

D. radiodurans Fapy and 8-oxoG DNA glycosylase activities in the presence of various concentrations of anti-Fpg antibodies. Fraction V (0.04 U of Fapy DNA glycosylase; ■) and fraction VI (0.04 U of Fapy DNA glycosylase; ▴) were incubated for 10 min at 32°C with 700 fmol of [3H]Fapy–poly(dG-dC) or 100 fmol of 8-oxoG/C-containing oligonucleotide (34-mer) (B).

FIG. 4

FIG. 4

Fapy-DNA glycosylase activities of fraction V (■) and fraction VI (▴). Increasing amounts of each enzyme were incubated in the presence of [3H]Fapy–poly(dG-dC) at 32°C for 10 min, and the [3H]Fapy residues released were measured (for details, see Materials and Methods).

FIG. 5

FIG. 5

Cleavage of DNA duplexes containing a single 8-oxoG mismatched with one of the four bases C (■), T (▾), G (▴), and A (•). The various duplexes (100 fmol) were incubated with increasing amounts of proteins standardized as Fapy-DNA glycosylase activity of fraction V (A) and fraction VI (B) for 10 min at 32°C.

FIG. 6

FIG. 6

NaBH4 trapping of 8-oxoG-containing DNA (200 fmol) treated with different amounts of proteins standardized as Fapy-DNA glycosylase activity from fraction V (■) and fraction VI (▴). The reactions were performed at 32°C for 30 min (for details, see Materials and Methods).

FIG. 7

FIG. 7

Cleavage of an oligonucleotide containing a unique abasic site. Oligonucleotide (100 fmol) was incubated for 10 min at 32°C with enzyme. The products of the reaction were separated by SDS-PAGE, and the results were quantified with PhosphorImager. (A) Increasing amounts of fraction V (■) and fraction VI (▴). (B) Lane 1, duplex oligonucleotide containing an AP site. lane 2, like lane 1 but incubated with 0.2 M NaOH before analysis; lane 3, like lane 1 but incubated with 10 ng of E. coli Fpg protein; lane 4, like lane 1 but incubated with 10 ng of E. coli Nth protein; lane 5, like lane I but incubated with 10 ng of fraction V enzyme; lane 6, like lane 1 but incubated with 10 ng of fraction VI enzyme.

FIG. 8

FIG. 8

NaBH4 trapping of AP site-containing DNA (100 fmol) with 50 ng of E. coli Fpg protein (lane 1), 10, 20 50 ng of fraction V enzyme (lanes 2, 3, and 4), and 10, 20, and 50 ng of fraction VI enzyme (lanes 5, 6, and 7) for 10 min at 32°C.

FIG. 9

FIG. 9

Incision of OsO4-treated DNA by the D. radiodurans enzymes. (A) 500 ng of OsO4-treated DNA was incubated at 32°C for 10 min with increasing amounts of fraction V (■) and fraction VI (▴) enzymes. The products of the reaction were separated on an agarose gel and quantified by microdensitometric analysis (for details, see Materials and Methods). (B) Thymine glycol DNA glycosylase activities of fraction V (■) and fraction VI (▴) enzymes in the presence of different dilutions of anti E. coli Fpg antibodies.

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