Title page for ETD etd-04032007-173430


Type of Document Dissertation
Author Bhattacharyya, Gargi
URN etd-04032007-173430
Title DNA-Binding and Oligomerization Properties of a Functionally Distinct Dps Homolog, Dps-1 from Deinococcus radiodurans
Degree Doctor of Philosophy (Ph.D.)
Department Biological Sciences
Advisory Committee
Advisor Name Title
Anne Grove Committee Chair
Grover L. Waldrop Committee Member
Patrick DeMario Committee Member
Yong-Hwan Lee Committee Member
Shisheng Li Dean's Representative
Keywords
  • dna-binding
  • metal-binding
  • emsa
  • dps-1
  • ferroxidation
  • oligomerization
Date of Defense 2007-03-29
Availability restricted
Abstract
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.

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