1. 1.DNA polymerase I has three enzymatic activities - a 5' to 3' DNA polymerase activity, a 3' to 5' exonuclease activity (also called proofreading), and a 5' to 3' exonuclease activity.
2. All DNA polymerases require a primer to start DNA synthesis. The primer is formed inside of cells by a special RNA polymerase known as primase. (RNA polymerase does not require a primer)
3. DNA replication proceeds by two distinct mechanisms (both 5'-3', however)- one on each strand. Leading strand and lagging strand synthesis occur by different mechanisms, but both are catalyzed by the same DNA replication complex (Pol III, in the case of E. coli).
4. Leading strand synthesis is continuous in the 5' to 3' direction. Lagging strand synthesis can only occur when the leading strand synthesis opens up a new single stranded region for replication. The 5' to 3' syntheses of the lagging strand are discontinuous. The many pieces of lagging strand synthesis are called Okazaki fragments.
5. Okazaki fragments must be combined together ultimately. First, the RNA primer must be removed from each one. The 5' to 3' exonuclease activity of DNA Polymerase I is needed to remove the initial RNA primer of leading strand synthesis, but is needed frequently to remove the primers of lagging strand synthesis.
6. DNA ligase is an enzyme that creates phosphodiester bonds between adjacent nucleotides between Okazaki fragments. Biotechnologists use this enzyme to join DNA fragments together to create recombinant molecules.
7. E. coli DNA replication occurs at 1000 base pairs per second. At 10 base pairs per turn, this represents a machine turning at 5000 to 6000 rpm. E. coli's helicase protein (DNA B - part of the BC complex) unwinds DNA at a rate of at least 5000 rpm. The protein separates strands ahead of the DNA Pol III so as to make single strands accessible for replication. Unwinding of strands causes superhelical tension to increase ahead of the helicase. Topoisomerase II (gyrase) relieves the tension created by the helicase and is essential for replication to proceed efficiently.
8. DNA Polymerase III is very processive in its action, meaning that once it gets onto a DNA molecule, it stays on it for a long time replicating it. DNA Polymerase I is NOT very processive.
9. In E. coli DNA replication, a dimer of DNA Polymerase III is at the replication fork and performs most of the DNA replication in the cell. One portion of it replicates the leading strand and the other replicates the lagging strand. Leading strand synthesis is faster, so the lagging strand template sometimes loops out in a trombone-like fashion when the lagging strand replication falls behind.
10. Proteins at/near the replication fork and their functions described so far include primase (makes RNA primers necessary for the DNA polymerase to act on), SSB (single stranded binding protein - protects single-stranded DNA and interacts with the replication proteins), DNA gyrase (topoisomerase II - relieves the superhelical tension created by helicase), Pol I (removes RNA primers), DNA ligase (joins DNA fragments together by catalyzing synthesis of phosphodiester bonds at nick sites), and helicase (unwinds double helix).
This course in general biochemistry is intended to integrate information about metabolic pathways with respiration (respiratory control) and initiate the student into a microscopic world where blueprints are made of deoxyribonucleic acids, factories operate using enzymes, and the exchange rate is in ATPs rather than Yens or Euros. Beyond explaining terms, and iterating reactions and metabolic pathways, this course strives to establish that the same principles that govern the behavior of the world around us also govern the transactions inside this microscopic world of the living cell. And by studying and applying these principles, we begin to understand cellular and bodily processes that include sensory mechanisms.
1. Lipids, Membranes and Transport
2. Electron Transport, Oxidative Phosphorylation and Mitochondrial 3. Transport Systems
3. Lipid Metabolism
4. Nucleotide Metabolism
5. DNA Replication