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T4 DNA polymerase. Rates and processivity on single-stranded DNA templates
Journal of Molecular Biology
|August 5, 1984
Summary
Bacteriophage T4 DNA polymerase synthesizes DNA at approximately 250 nucleotides per second in vitro. This rate is limited by enzyme processivity and DNA binding, with only a fraction of molecules active at any time.
Area of Science:
- Molecular Biology
- Virology
- Biochemistry
Background:
- Bacteriophage T4 DNA polymerase is crucial for viral DNA replication.
- Understanding the enzyme's kinetics is essential for elucidating DNA synthesis mechanisms.
Purpose of the Study:
- To determine the in vitro DNA synthesis rate of individual bacteriophage T4 DNA polymerase molecules.
- To compare the in vitro enzymatic rate with in vivo replication fork progression.
Main Methods:
- Utilized three distinct experimental methods to measure DNA polymerase movement.
- Assessed enzyme activity on a single-stranded DNA template at 37 degrees C.
Main Results:
- The in vitro rate of bacteriophage T4 DNA polymerase was determined to be approximately 250 nucleotides per second.
- This rate closely approximates the in vivo replication fork speed of bacteriophage T4 (around 500 nucleotides per second).
- A small proportion of T4 DNA polymerase molecules were actively synthesizing DNA in vitro.
Conclusions:
- The limited processivity and non-productive DNA binding of T4 DNA polymerase explain the low fraction of active molecules.
- The in vitro enzymatic rate provides valuable insights into the speed of viral DNA replication.