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Direct observation of single MuB polymers: evidence for a DNA-dependent conformational change for generating an
Eric C Greene1, Kiyoshi Mizuuchi
1Laboratory of Molecular Biology, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
Abstract:
MuB, an ATP-dependent DNA binding protein, is critical for selection of target sites on the host chromosome during Mu transposition. We have developed a system for observing the behavior of single MuB polymers bound to an immobilized molecule of DNA. We show that the individual polymers display a broad distribution of disassembly rates and exhibit regional variations in DNA binding. Additionally, ATP hydrolysis was obligatorily coupled to dissociation of MuB subunits from the DNA during polymer disassembly. We propose a model in which the formation of an active target complex is mediated by a conformational change within the MuB polymer that is influenced by the sequence of the DNA.
Insights
Single MuB protein polymers show varied DNA binding and disassembly rates. ATP hydrolysis drives MuB subunit dissociation, suggesting DNA sequence influences target complex formation.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- MuB protein is essential for site selection in Mu transposition.
- Understanding MuB's DNA binding and dynamics is key to Mu transposition mechanisms.
Purpose of the Study:
- To observe the behavior of single MuB polymers on immobilized DNA.
- To elucidate the role of ATP hydrolysis in MuB polymer disassembly.
- To investigate the influence of DNA sequence on MuB binding and target complex formation.
Main Methods:
- Developed a single-molecule observation system for MuB-DNA interactions.
- Analyzed the disassembly rates of individual MuB polymers.
- Studied the coupling of ATP hydrolysis to MuB subunit dissociation.
Main Results:
- MuB polymers exhibit heterogeneous disassembly rates and regional DNA binding variations.
- ATP hydrolysis is mandatory for MuB subunit dissociation from DNA.
- DNA sequence influences MuB polymer conformational changes and binding.
Conclusions:
- MuB polymer dynamics are complex and sequence-dependent.
- A model is proposed where DNA sequence-mediated conformational changes in MuB polymers facilitate target complex formation during transposition.
