Phosphorothioated DNA Is Shielded from Oxidative Damage.

Area of Science:

• Biochemistry
• Molecular Biology
• Genetics

Background:

• DNA phosphorothioation (PT) is a modification where an oxygen atom on the DNA backbone is replaced with a sulfur atom.
• PT modification can lead to genomic instability under oxidative stress but confers resistance to hydrogen peroxide (H2O2) in catalase-deficient strains.
• The physiological role of DNA PT modification in bacteria across diverse environments remains largely unknown.

Purpose of the Study:

• To biochemically characterize the purified PT modification protein complex (DndCDE) from *S. enterica*.
• To elucidate the mechanism by which PT modification confers H2O2 resistance.
• To investigate the enzymatic activity and DNA-binding properties of the DndCDE complex.

Main Methods:

• Native polyacrylamide gel electrophoresis (PAGE) to determine oligomeric states of DndCDE.
• Biochemical assays to measure H2O2 decomposition activity of DndCDE-FeS.
• Analysis of the role of the iron-sulfur (Fe-S) cluster and cysteine residues in protein activity.
• Detection of catalase activity in DndCDE from *Pseudomonas fluorescens*.

Main Results:

• The DndCDE protein complex exists in multiple oligomeric states and binds avidly to PT-modified DNA.
• DndCDE-FeS exhibits H2O2 decomposition activity (Vmax = 10.58 mM min-1, K0.5S = 31.03 mM, Hill coefficient = 2.42).
• Catalase activity is dependent on an intact DndCDE complex and an iron-sulfur cluster on the DndC subunit, with additional cysteine residues playing a catalytic role.
• Catalase activity was also observed in DndCDE from *P. fluorescens*.

Conclusions:

• DndCDE-FeS functions as a short-lived catalase.
• The DndCDE complex binds to PT sites on DNA, forming a protective 'catalase shield' against H2O2 damage.
• This mechanism potentially explains the paradox of PT modification conferring H2O2 resistance despite increased susceptibility to oxidative cleavage.