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Regulation of T7 gp2.5 binding dynamics by its C-terminal tail, template conformation and sequence
Longfu Xu1, Jordi Cabanas-Danés1, Matthew T J Halma1
1Department of Physics and Astronomy, Vrije Universiteit Amsterdam, De Boelelaan 1081, 1081 HV, Amsterdam, The Netherlands.
Nucleic Acids Research
|May 31, 2023
Summary
Bacteriophage T7 DNA-binding protein (gp2.5) binds single-stranded DNA, preventing secondary structures. Its unique recycling behavior ensures efficient DNA replication during Okazaki fragment synthesis.
Area of Science:
- Molecular Biology
- Biophysics
Background:
- Bacteriophage T7 single-stranded DNA-binding protein (gp2.5) is crucial for DNA replication.
- It interacts with replication proteins and protects exposed single-stranded DNA (ssDNA).
Purpose of the Study:
- To visualize and characterize the single-molecule binding dynamics of T7 gp2.5 to ssDNA.
- To understand how T7 gp2.5 suppresses secondary structure formation and facilitates DNA replication.
Main Methods:
- Single-molecule visualization of fluorescently labeled T7 gp2.5 binding to ssDNA.
- Investigating binding dynamics under varying template tensions using a deletion mutant (gp2.5-Δ21C).
Main Results:
- T7 gp2.5 reduces ssDNA contour length in a force-dependent manner, suppressing secondary structures.
- DNA base sequence, ssDNA conformation, and the protein's C-terminal domain influence binding parameters.
- A template-catalyzed recycling behavior leads to apparent cooperative binding and efficient redistribution.
Conclusions:
- T7 gp2.5 employs an efficient mechanism to prevent ssDNA secondary structures during replication.
- Rapid rebinding to exposed ssDNA regions facilitates Okazaki fragment synthesis and spatial redistribution.
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