DNA-Binding and Oligomerization Properties of a Functionally Distinct Dps Homolog, Dps-1 from Deinococcus radiodurans
Dps (DNA protection during starvation) proteins play an important role in the protection of prokaryotic macromolecules from damage by reactive oxygen species. The Dps homolog, Dps-1, from the radiation-resistant bacterium Deinococcus radiodurans has an extended N-terminal tail. In the crystal structure of Dps-1, the first ~30 N-terminal residues are invisible and the remaining 25 residues form a loop that harbors a novel metal binding site. The data presented here show that retention of this N-terminal metal site is necessary for formation of the dodecameric protein assembly. Previous studies have suggested that the lysine-rich N-terminus of Dps proteins participates in DNA binding. Accordingly, deletion of the N-terminal tail of Dps-1 obliterates DNA/Dps-1 interaction. Electrophoretic mobility shift assays using DNA modified with specific major/minor groove reagents show that Dps-1 interacts through the DNA major groove. Dodecameric Dps-1 can bind ? 22bp DNA duplexes with very high affinity (Kd ~0.4 nM); considering interactions in the DNA major grooves, the requirement for two complete helical turns implies optimal interactions involving two consecutive major grooves. The data further suggests that high-affinity DNA binding depends on occupancy of the N-terminal metal site. Stoichiometric titration of dodecameric Dps-1 with 22 bp DNA revealed the presence of 6 DNA binding sites in each dodecamer. DNA cyclization assays show that dodecameric Dps-1 inhibits DNA bending. Taken together, the mode of DNA interaction by Dps-1 is consistent with the previously proposed layered assembly of protein and DNA that leads to DNA compaction. Using Dps-1-promoter-lacZ fusion constructs, it is shown that Dps-1 expression in D. radiodurans is relatively constant throughout both exponential and stationary phase growth. As E. coli cells expressing Dps-1 feature significant nucleoid condensation, as shown by transmission electron microscopy and nucleoid staining, a role for Dps-1 in chromosomal DNA packaging is suggested. The presence of a novel iron exit channel is most likely responsible for the inability of Dps-1 to protect DNA from hydroxyl radical-mediated DNA degradation. The release of iron from the core upon DNA binding suggests that Dps-1 may be involved in the process of DNA degradation that contributes the first response to DNA damage.
Advisor:Grover L. Waldrop; Patrick DeMario; Yong-Hwan Lee; Shisheng Li; Anne Grove
School:Louisiana State University in Shreveport
School Location:USA - Louisiana
Source Type:Master's Thesis
Date of Publication:04/09/2007