Highlights Lecture #16 Spring 2017

Highlights DNA Replication II

1. Replication of each strand of a DNA duplex occurs by a different scheme. The leading strand is made in a single continuous piece. The lagging strand is made in short segments called Okazaki fragments. The two different replication strategies arise from the fact that both must occur in the 5′ to 3′ direction. The lagging strand segments can only be started after the leading strand synthesis opens up the duplex sufficiently. This occurs repetitively during synthesis, generating multiple lagging strand fragments.

2. Note that 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.

3. 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.

4. All DNA polymerases (enzymes which catalyze the synthesis of DNA) require 1) 4 dNTPs – dATP, dTTP, dGTP, dCTP; 2) a primer which they can extend; and 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.

5. DNA replication always starts at a specific sequence called an origin of replication (also called ori). Prokaryotic cells have one origin of replication per chromosome. Eukaryotic cells have many origins of replication per chromosome.

6. DNA Polymerase I contains three enzymatic activities – 5′ to 3′ polymerization (makes phosphodiester bonds that make DNA strands), 3′ to 5′ exonuclease (remove mismatch errors that occur during replication – also called proofreading), and 5′ to 3′ exonuclease (removes RNA primers). DNA Polymerase III lacks the 5′ to 3′ exonuclease.

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. The beta clamp is a protein that forms a loop around DNA and “holds” the DNA Polymerase III to the DNA so it does not fall off. DNA Polymerase I is NOT held by the beta clamp and falls off frequently.

9. DNA replication in eukaryotes is problematic at the very ends of the linear chromosomes. There the RNA primers CANNOT be replaced by DNA, meaning that each time they get replicated, a small piece of their DNA is lost. An older cell will have shorter sequences at the end of its chromosomes than a younger cell will.

10. Eukaryotic chromosomes have sequences called telomeres at the ends of the chromosomes. These are small sequences (7-10 nucleotides) that are repeated thousands of times.

11. Telmomeres are made during embryonic development by an enzyme called telomerase that carries its own template.

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