Module 02 - Section 01 Flashcards
Beginnings of replication enzymology
What are the 5 phases of the Eukaryotic Cell cycle?
G0 Phase, G1 Phase, S Phase, G2 Phase, M Phase
Which phases of the cell cycle - cells are not actively dividing
G0, G1
Which phase is not technically part of the cell cycle
G0
First growth phase of cell cycle
G1
Which phase of cell cycle; cell replicates its entire genome
S phase
Cell cycle; 2nd growth phase
G2
Cell cycle: cell division occurs
M phase
Describe # of chromosome in G1 phase
2n: cells are diploid containing two copies of each chromosome
Describe chromosome in S phase
DNA replication is occurring - chromosomes are duplicated
Describe # of chromosome in G2 phase
4n: cells are tetraploid, containing four copies of each chromosome
Describe # of chromosome in M phase
2n: each daughter cells contains a complete set of chromosomes
Arthur Kornberg discoveries (3)
(1) assay for measuring the amount of DNA synthesis that is occurring
(2) Several heat-stable factors are needed for the DNA synthesis reaction (nucleoside triphosphates)
(3) DNA polymerase I purification using multi-step fractionation of the bacterial lysate
Describe briefly the Purification of Polymerase by Kornberg
Step 1: break the bacteria by sonication
Step 2 to 5: remove DNA and RNA from the mixture
Step 6: use salt to precipitate some proteins and leave others in the solution (based on their proteins)
Step 7: use ion exchange chromatography to give a more purified product
What is sonication?
Applying sound energy to agitate particles in a sample
Describe Kornberg’s experiments for requirements for polymerization reaction
Determined the maximal incorporation of deoxyribonucleotides into DNA under various conditions. They performed 8 experiments, each one removing one or more factors and radiolabelling dTTP to monitor the effect on incorporation
What were the results of Kornberg’s requirements of the polymerization reaction results?
In each experiment where a factor was removed, there was almost complete loss of the incorporation of radiolabelled dTTP into DNA
What do the results of Kornberg’s requirements mean for the polymerization reaction?
The requirements of the polymerase reaction are:
- all 4 nucleotides present in the cell
- an intact DNA template
- a Mg++ cofactor for the polymerase
