How did O'Donnell show that the sliding clamp complex actually slides along dsDNA providing processivity to DNA Pol III?
I think he took dna, a primer, and one radioactive dNTPs, added Pol III that was isolated, ran a gel, and the conclusion was that it was processive because on the gel, the distributive section had many bands and processive had one band. I dunno. does anyone know what the experiment was called and what the hypothesis was? Please, I want serious answers
The experiment you are referring to is known as the "Clamp Loader Assay". It was performed by O'Donnell and colleagues to demonstrate the sliding mechanism of the sliding clamp complex in providing processivity to DNA polymerase III (Pol III).
The hypothesis of the experiment was that the sliding clamp complex moves along the DNA template, allowing DNA Pol III to remain associated with the DNA strand and synthesize DNA continuously without dissociating after each round of synthesis.
Here is a step-by-step breakdown of the experiment:
1. Preparation: O'Donnell and his team used a DNA template strand, a short primer, and radioactive labeled dNTPs to monitor the synthesis of DNA.
2. Isolation of DNA Pol III: Pol III was isolated from the bacteria Escherichia coli. This enzyme is responsible for DNA replication in bacteria and is highly processive, meaning it can synthesize DNA continuously without falling off the template strand.
3. Binding of the sliding clamp complex: The sliding clamp complex, also known as the beta clamp, was added to the DNA template strand. This clamp encircles the DNA and helps to stabilize the DNA Pol III-DNA interaction, enhancing the polymerase's processivity.
4. Polymerase reaction: The reaction mixture contained all the components necessary for DNA synthesis, including the DNA template strand, the primer, the radioactive dNTPs, and the DNA Pol III with the sliding clamp complex attached.
5. Gel electrophoresis: After the incubation period, the reaction mixture was loaded onto an agarose gel. Gel electrophoresis was performed to separate the DNA fragments based on their size.
6. Analysis of results: The gel was then exposed to X-ray film to visualize the radioactive bands corresponding to the synthesized DNA fragments. The radioactive bands represent the newly synthesized DNA.
The conclusion of the experiment was based on the observation that in the absence of the sliding clamp complex, DNA Pol III exhibited distributive synthesis, resulting in multiple bands of different lengths on the gel. However, when the sliding clamp complex was present, there was only a single band observed at a higher molecular weight. This single band indicated processive synthesis, as the polymerase continuously synthesized DNA without dissociation.
Through this experiment, O'Donnell demonstrated that the sliding clamp complex allows DNA Pol III to slide along the DNA template, enabling continuous DNA synthesis and conferring processivity to the polymerase.