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Purification and characterization of the coliphage N4-coded single-stranded DNA binding protein
G Lindberg1, S C Kowalczykowski, J K Rist
1Department of Biochemistry, University of Chicago, Illinois 60637.
The Journal of Biological Chemistry
|July 25, 1989
Summary
Researchers purified and characterized N4 single-stranded DNA binding protein (N4 SSB), finding it binds cooperatively to ssDNA and enhances N4 DNA polymerase activity by increasing processivity and melting hairpins.
Area of Science:
- Molecular Biology
- Virology
- Biochemistry
Background:
- Coliphage N4 infection induces specific proteins.
- Single-stranded DNA binding proteins play crucial roles in DNA metabolism.
Purpose of the Study:
- To purify and characterize the N4 single-stranded DNA binding protein (N4 SSB).
- To investigate the binding properties and functional roles of N4 SSB.
Main Methods:
- Protein purification and characterization.
- Fluorescence quenching assay to quantify DNA binding.
- Analysis of binding affinity, cooperativity, and salt dependence.
- Melting point determination of DNA duplexes.
- Assay of N4 SSB's effect on N4 DNA polymerase activity.
Main Results:
- N4 SSB is a 31 kDa protein with 10 tyrosine residues, crucial for its fluorescence.
- The protein binds single-stranded DNA (ssDNA) cooperatively, with higher affinity for DNA than RNA.
- Binding is salt-dependent, involving displacement of ions, and exhibits a cooperativity factor (omega) of 300.
- N4 SSB significantly lowers the melting point of poly(dA.dT).poly(dA-dT) but not natural DNA.
- N4 SSB enhances N4 DNA polymerase processivity and overcomes polymerization-blocking hairpin structures.
Conclusions:
- N4 SSB is a key viral protein involved in DNA replication.
- Its cooperative binding and ability to modulate DNA structure facilitate viral DNA synthesis.