Highlights DNA Synthesis II

1.E. coli DNA Polymerase I (also called Pol I) functions in repair of DNA and in removal of RNA primers. The latter is the most essential function of the enzyme. Pol I and DNA Polymerase III Holoenzyme (also called Pol III) both have an ability to correct mismatch errors made during polymerization. This activity is called 'proofreading' and is important for improving the accuracy (fidelity) of DNA replication. The proofreading activity results in the removal of mispaired bases in the DNA as it is being made. The enzymatic activity of the enzyme that performs the proofreading is known as the 3' to 5' activity.

2. Analogs of nucleosides are used medicinally to stop DNA replication of certain viruses. They include AZT and ddI, which are both used to treat HIV. They act by stopping the elongation of DNA by reverse transcriptase.

Enzymes that change the "twisting" of DNA are called topoisomerases. One example is E. coli DNA Gyrase, which acts during E. coli DNA replication. It acts in replication to 'untangle' the super-twisted DNA created during the unwinding of strands at the replication fork.

4. Replication of DNA occurs at a replication fork. The replication fork includes several proteins that help DNA replication to occur. The most interesting of these is the helicase which separated DNA strands and (in E. coli) operates at about 6000 RPM. Unwinding of strands generates superhelical tension ahead of the site of unwinding. This superhelical tension is relieved in E. coli by a topoisomerase known as DNA gyrase.

5. Prokaryotic replication forks are bidirectional, having started from a single replication origin in opposite directions. The major players in E. coli replication are - 1) DNA polymerase III complex; 2) beta clamp (holds polymerase complex 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.

6. 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.

7. Proofreading improves the accuracy of DNA replication about a thousand fold. Cells that have mutations that destroy the proofreading of the DNA polymerases form mutations at a much higher rate than normal cells.

8. 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 remove 8-oxo-guanine from DNA. Base excision repair is a cellular process for removing damaged based in a DNA strand. This occurs as a result of action by an enzyme which catalyzes the removal of the damaged base followed by an exonuclease action to remove a few more bases and then filling in of the gap by DNA polymerase(s) and sealing of the last bond by DNA ligase.

9. Another repair mechanism in E. coli is that of the Mut S / Mut H / Mut L system, which recognizes a mismatch that occurs in DNA as a result of an error in polymerization and proofreading.

10. 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.

11. Nucleotide excision repair is a system used to repair thymine dimers (among other problems). In this method, a stretch of DNA containing the damaged bases is removed and DNA polymerase(s) fill in the gap, followed by ligation by DNA ligase to finish the process.