The N-terminal Extensions of Deinococcus radiodurans Dps-1 Mediate DNA Major Groove Interactions as well as Assembly of the Dodecamer

Anne Grove,Gargi Bhattacharyya

doi:10.1074/jbc.m611255200

Abstract

Dps (DNA protection during starvation) proteins play an important role in the protection of prokaryotic macromolecules from damage by reactive oxygen species. Previous studies have suggested that the lysine-rich N-terminal tail of Dps proteins participates in DNA binding. In comparison with other Dps proteins, Dps-1 from Deinococcus radiodurans has an extended N terminus comprising 55 amino acids preceding the first helix of the 4-helix bundle monomer. In the crystal structure of Dps-1, the first approximately 30 N-terminal residues are invisible, and the remaining 25 residues form a loop that harbors a novel metal-binding site. We show here that deletion of the flexible N-terminal tail obliterates DNA/Dps-1 interaction. Surprisingly, deletion of the entire N terminus also abolishes dodecameric assembly of the protein. Retention of the N-terminal metal site is necessary for formation of the dodecamer, and metal binding at this site facilitates oligomerization of the protein. Electrophoretic mobility shift assays using DNA modified with specific major/minor groove reagents further show that Dps-1 interacts through the DNA major groove. DNA cyclization assays suggest that dodecameric Dps-1 does not wrap DNA about itself. A significant decrease in DNA binding affinity accompanies a reduction in duplex length from 22 to 18 bp, but only for dodecameric Dps-1. Our data further suggest that high affinity DNA binding depends on occupancy of the N-terminal metal site. Taken together, the mode of DNA interaction by dodecameric Dps-1 suggests interaction of two metal-anchored N-terminal tails in successive DNA major grooves, leading to DNA compaction by formation of stacked protein-DNA layers.

Highlights

Cellular macromolecules, including proteins, lipids, and DNA
The mesophilic, non-spore-forming eubacterium Deinococcus radiodurans is known for its extraordinary ability to withstand the lethal and mutagenic effects of DNA damaging agents, such as ionizing radiation and desiccation, both conditions that are characterized by the presence of oxidative radi
The effect of netropsin on TATA box binding-protein (TBP)-DNA complex formation was studied in parallel where the same 26-bp duplex DNA was incubated with 1070 pmol of TBP in a reaction buffer (40 mM Tris-HCl, pH 8.0, 10 mM NaCl, 7 mM MgCl2, 3 mM dithiothreitol, 10 ␮g/ml BSA) followed by the addition of netropsin

Summary

Introduction

Cellular macromolecules, including proteins, lipids, and DNA. they have developed a number of defense mechanisms to combat such stress conditions in the environment. Netropsin Assay—Eight nM dodecameric Dps-1 was incubated with 50 fmol of 26-bp double-stranded 32P-labeled DNA containing an 8-bp TATA box (the sequence available in the supplemental material) in 10 ␮l of binding buffer with 500 mM NaCl at room temperature for 45 min.

Results

Conclusion