Related Experiment Videos
Crystal structure of a prokaryotic replication initiator protein bound to DNA at 2.6 A resolution
H Komori1, F Matsunaga, Y Higuchi
1Department of Chemistry, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto 606-8502, USA.
Abstract:
The initiator protein (RepE) of F factor, a plasmid involved in sexual conjugation in Escherichia coli, has dual functions during the initiation of DNA replication which are determined by whether it exists as a dimer or as a monomer. A RepE monomer functions as a replication initiator, but a RepE dimer functions as an autogenous repressor. We have solved the crystal structure of the RepE monomer bound to an iteron DNA sequence of the replication origin of plasmid F. The RepE monomer consists of topologically similar N- and C-terminal domains related to each other by internal pseudo 2-fold symmetry, despite the lack of amino acid similarities between the domains. Both domains bind to the two major grooves of the iteron (19 bp) with different binding affinities. The C-terminal domain plays the leading role in this binding, while the N-terminal domain has an additional role in RepE dimerization. The structure also suggests that superhelical DNA induced at the origin of plasmid F by four RepEs and one HU dimer has an essential role in the initiation of DNA replication.
Insights
The RepE initiator protein controls DNA replication in E. coli by acting as a monomer initiator or dimer repressor. Its crystal structure reveals how RepE domains bind DNA and suggests supercoiled DNA is key for replication initiation.
Area of Science:
- Molecular Biology
- Structural Biology
- Microbiology
Background:
- The F factor plasmid in Escherichia coli facilitates sexual conjugation.
- Initiator protein RepE has dual roles in DNA replication initiation: monomer as initiator, dimer as repressor.
Purpose of the Study:
- To determine the crystal structure of the RepE monomer bound to the F plasmid's iteron DNA sequence.
- To elucidate the mechanism of RepE's DNA binding and its role in replication initiation.
Main Methods:
- X-ray crystallography to solve the RepE monomer-DNA complex structure.
- Structural analysis to understand protein-DNA interactions and dimerization interfaces.
Main Results:
- The RepE monomer structure shows pseudo 2-fold symmetry between N- and C-terminal domains.
- Both domains bind the iteron DNA major grooves, with the C-terminal domain being primary for binding.
- The N-terminal domain contributes to RepE dimerization, and the structure suggests supercoiled DNA is crucial for initiation.
Conclusions:
- RepE's dual function is structurally explained by its monomeric and dimeric states.
- The detailed structure provides insights into plasmid DNA replication control in E. coli.
- Superhelical DNA formation is implicated as essential for initiating F factor replication.