Kevin Ahern's BB 350 (DNA Replication II) 2014 - #16

1. Contact me at [email protected] / Friend me on Facebook (kevin.g.ahern) 2. Download my free biochemistry book at http://biochem.science.oregonstate.ed... 3. Take my free iTunes U course at https://itunes.apple.com/us/course/bi... 4. Check out my free book for pre-meds at http://biochem.science.oregonstate.ed... 5. Lecturio videos for medical students - https://www.lecturio.com/medical-cour... 6. Course video channel at http://www.youtube.com/user/oharow/vi... 7. Check out all of my free workshops at http://oregonstate.edu/dept/biochem/a... 8. Check out my Metabolic Melodies at http://www.davincipress.com/ 9. My courses can be taken for credit (wherever you live) via OSU's ecampus. For details, see http://ecampus.oregonstate.edu/soc/ec... 10. Course materials at http://oregonstate.edu/instruct/bb350 Highlights DNA Synthesis 1. Leading AND lagging strand synthesis are both occurring at the same replication fork AND both are occurring exclusively in the 5' to 3' direction. 2. All DNA polymerases require 1) 4 dNTPs - dATP, dTTP, dGTP, dCTP; 2) a primer which they can extend; 3) a template (complementary strand) they can copy. All DNA polymerases make DNA in the 5' to 3' direction. Primers for DNA replication in cells are short RNAs that are made by an enzyme called Primase. 3. Note that both leading AND lagging strand synthesis are both occurring at the same replication fork AND that both leading and lagging strand synthesis are occurring exclusively in the 5' to 3' direction. 4. E. coli cells have five DNA polymerases in them. DNA polymerase I (Pol I) was the first one discovered (by Arthur Kornberg in the late 1950s). Pol I does not polymerize DNA fast enough to complete replication of the E. coli chromosome (6,000,000 base pairs) in the 20-25 minutes it is known to occur in. 5. The enzyme responsible for replicating most of the E. coli chromosome is DNA polymerase III. It is capable of synthesizing 1000 base pairs per second. 6. Prokaryotic replication forks are bidirectional, having started from a single replication origin (the origin is where replication starts) in opposite directions. The major players in E. coli replication are 1) DNA polymerase III; 2) beta clamp (holds polymerase to DNA); 3) Single strand binding protein - protects single strand DNA; 4) helicase - unwraps DNA duplex ahead of replication fork; 5) primase - makes RNA primer necessary to start DNA replication; 6) DNA gyrase - topoisomerase that relieves superhelical tension created by helicase; 7) DNA ligase - joins pieces of DNA, such as Okazaki fragments together; 8) DNA polymerase I - removes RNA primers and replaces with DNA. 7. Proofreading is a phenomenon that occurs as a result of a 3' to 5' exonuclease activity on many DNA polymerases (including DNA Polymerases I and III). Proofreading improves the accuracy of DNA replication about a hundred fold. 8. The most prominent DNA polymerases that lack proofreading are those of reverse transcriptases of retroviruses. HIV's reverse transcriptase is VERY error prone and is the primary reason that drugs lose the effectiveness on the disease over time. 9. DNA Polymerase III is a multi-subunit enzyme that gets on a DNA stays on it for a long time (highly processive). DNA Polymerase I is not so highly processive. The difference in their processivity is the fact that Polymerase III has the beta clamp, which is a ring that helps hold it tightly to DNA during replication. 10. Ultraviolet light can act as a mutagen (an agent that causes mutations). It does this by creating cross-links between adjacent thymines in a strand of DNA. If these are not properly repaired, a mutation can result. It is for this reason that people who tan/burn in tanning booths often develop skin cancer later in life. Repairing this damage is performed by the system known as nucleotide excision repair. 11. Thymine dimers are probably the most common damage that occurs to DNA. They arise from exposure to UV light (tanning booths, excessive sun tanning) and result in a covalent bond formed between adjacent thymine bases in DNA. If they are not repaired, thymine dimers can result in mutation. The more exposure you have to UV light, the more likely you will develop skin cancer. Stay out of tanning booths. 12. DNA can be damaged by other means. Oxidation is a common one. When oxidation of guanine occurs, for example, 8-oxo-guanine is created. This base can form stable base pairs with adenine. If DNA containing 8-oxo-guanine is allowed to replicate, adenine will be inserted in place of cytosine, causing a mutation. A repair mechanism called base excision repair helps to fix this. 13. Another repair mechanism in E. coli is that of mismatch repairthat occurs in DNA as a result of an error in polymerization and proofreading.