Lecture 06 – DNA ReplicationBased on Chapter 3 DNA Replication

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1. Semiconservative DNA Replication

2a. The Meselson–Stahl Experiment

2b. The Meselson–Stahl Experiment

2c. The Meselson–Stahl Experiment

3. DNA Polymerase (Kornberg Enzyme)4. DNA Polymerase I

• First isolation of an enzyme involved in DNA replication was in 1955. Arthur Kornberg won the 1959 Nobel Prize in Physiology or Medicine for this work.

• Kornberg isolated DNA polymerase I.• Availability of DNA Pol I allowed characterization of DNA

synthesis in vitro. Five components are needed:– Radioactively labeled dNTPs (all four: dATP, dGTP, dTTP,

and dCTP).– DNA polymerase I.– E. coli DNA (template).– Primer DNA.– Magnesium ions (Mg++).

5. Roles of DNA Polymerases

6. Molecular Model of DNA Replication

7. Initiation of Replication

8a. Semidiscontinuous DNA Replication

8b. Semidiscontinuous DNA Replication

8c. Semidiscontinuous DNA Replication

8c. Semidiscontinuous DNA Replication

8d. Semidiscontinuous DNA Replication

8e. Semidiscontinuous DNA Replication

9. DNA Replication in Eukaryotes

10. Replicons

DNA replication is very similar in both prokaryotes and eukaryotes, except that eukaryotes have more than one chromosome.

11. Initiation of Replication

12. Eukaryotic Replication Enzymes

• Enzymes of eukaryotic DNA replication aren’t as well characterized as their prokaryotic counterparts.

• Fifteen DNA polymerases are known in mammalian cells:• Three DNA polymerases are used to replicate nuclear DNA.

– Pol a (alpha) extends the 10-nt RNA primer by about 30 nt.– Pol d (delta) and Pol ´ (epsilon) extend the RNA/DNA

primers, one on the leading strand and the other on the lagging (it is not clear which synthesizes which).

• Other DNA Pols replicate mitochondrial or chloroplast DNA, or they are used in DNA repair.

• Primer removal differs from that in prokaryotes. – Pol d continues extension of the newer Okazaki fragment,

displacing the RNA and producing a flap that is removed by nucleases, thus allowing the Okazaki fragments to be joined by DNA ligase.

13a. Replicating the Ends of Chromosomes

13b. Replicating the Ends of Chromosomes

• Most eukaryotic chromosomes have short, tandemly repeated sequences at their telomeres

• Telomerasecontains both protein and RNA • 11-bp RNA sequence used to synthesize

the new telomere repeat DNA. • Telomerase functions as reverse

transcriptase.• After telomerase adds telomere sequences,

chromosomal replication proceeds in the usual way.

• Telomere length may vary• Loss of telomerase activity results in limited

rounds of cell division before the cell death.

14. Assembling Newly Replicated DNA into Nucleosomes