Section 1: The beginnings of replication enzymology Flashcards
What are the 4 stages of the cell cyle
G0/G1
S
G2
M
What happens during each stage of the cell cycle
1) G0/G1-Cells aren’t dividing during this phase, it is the first growth phase of the cell cycle. G0 is also not part of the cell cycle but occurs when cells aren’t dividing
2) S phase-The cell replicates its entire genome in preparation for division
3) G2-The second growth phase of the cell cycle. The last chance for cells to grow before division
4) M Phase-Mitosis-Cell division occurs to give 2 identical daughter cells
What did Arthur Kornberg discover?
1) An assay for measuring the amount of DNA synthesis occurring using bacterial cell extracts, they are prepared from lysing bacteria to release all of the contents
Anassayis an investigative (analytic) procedure in laboratory medicine
2) They discovered several heat stable factors that weren’t proteins (which are usually denatured at high temps) that are needed for DNA synthesis. These are nucleoside triphosphates
3) He used a multi-step fractionation of the bacterial lysate (ruptured bacteria contents), and they purified the protein known as DNA pol 1
What are the steps that were used in the purification of polymerase
Step 1 Sonication-Breaking the bacteria with sonication
Step 2-5-Removed DNA and RNA from the mix
Step 6-Used salt (ammonium sulfate) to precipitate some proteins and leave others in solution based on properties
Step 7-Ion exchange chromatography
At each stage they measure the protein content with spectroscopy, SDS-PAGE, and other methods to see the concentrations in soln. They also measured the specific activity of various fractions meaning the ability of the given enzyme to perform polymerization. This specific activity (polymerization) increased as the protein got more purified.
What was monitored during the purification of polymerase?
Multistep process in which protein concentration and DNA pol activity were monitored. They purified a bacterial lysate and monitored the activity of the proteins in fractions for their ability to polymerize DNA.
THEY USED MULTIPLE STEPS TO PURIFY POLYMERASE, IT GOT PURER EVERY TIME AND THE SPECIFIC ACTIVITY OF THE BACTERIAL LYSATE INCREASED WITH EVERY PURIFICATION
How did Kornberg discover the requirements of the polymerization reaction?
THEY TRIED A VARIETY OF COMBINATIONS (8 TRIALS) TO FIND THE CRITICAL COMPONENTS FOR DNA REPLICATION. RADIOLABBLED DTTPs WERE USED TO OBSERVE THE ABILITY TO REPLICATE OF THE VARIOUS MIXTURES. THEY FOUND THAT 3 THINGS ARE NEEDED: 1) ALL 4 NUCLEOTIDES 2) AN INTACT DNA TEMPLATE 3) AN MG2+ COFACTOR FOR THE POLYMERASE
What are the necessary components to synthesize DNA?
All 4 nucleotides, an intact DNA template, or an MG 2+ cofactor
Q: How did Kornberg deduce the requirements of the polymerase reaction:
He observed that when he removed critical components of replication there was a near complete loss of enzyme activity as measured by the amount of radiolabelled dTTPs being incorporated into DNA.
What was the key replication component that Kornberg purified
DNA Pol 1
