V. cDNA Libraries (converting mRNA into “complementary DNA”

1. V. cDNA Libraries (converting mRNA into “complementary DNA” Removes the RNA part of RNA:DNA hybrids

2. Nov. 11, 2005 MBB 407/511 Lecture 19: Prokaryotic DNA Replication (Part I)

3. Landmark Experiments in DNA Replication Requirements of ALL DNA Polymerases Mechanism of DNA Replication DNA Polymerase I of E. coli and its activities

4. I. Why Study DNA Replication? 1) To understand cancer—uncontrolled cell division (DNA replication) 2) To understand aging—cells capable of finite # of doublings 3) To understand diseases related to DNA repair Example of premature aging NOT caused by a hereditary disease a) Bloom’s Syndrome b) Xeroderma Pigmentosum c) Werner’s Syndrome Keith Richards (of the Rolling Stones)

5. II. Landmarks in the Study of DNA Replication A. 1953 Watson and Crick: From the structure of DNA they predicted that the DNA strands could act as templates for the synthesis of new strands: base complementarity B. 1958 Meselson and Stahl Three Potential DNA Replication Models Old DNA New DNA

6. The Meselson-Stahl Experiment “The most beautiful experiment in biology” Parental Conclusion: DNA is Replicated Semiconservatively: 1. The parental strands separate during DNA replication. 2. Daughter DNA molecules consist of one new and one old (parental) strand.

7. III. General Features of DNA Replication All DNA Polymerases: 1. require a DNA template and a primer with a 3’ OH end (DNA polymerases can only elongate; no de novo initiation of DNA synthesis) Short RNA primers are needed for initiation in vivo 2. require dNTPs 3. synthesize DNA in a 5’ to 3’ direction. 4. require metal ions (Mg2+ or Zn2+) as cofactors divalent The Substrates for DNA Replication

8. 1 1 2 2 The Mechanism of DNA Synthesis DNA Synthesis Is Exergonic dNTP + (dXMP)n (dXMP)n+1 + P~P DG = -3.5 kcal/mole P~P  2 P DG = -7 kcal/mole Total: dNTP + (dXMP)n (dXMP)n+1 + 2 P DG = -10.5 kcal/mole

9. The Role of Metal Ions In DNA Synthesis

10. Steric Constraints Prevent Catalysis of rNTPs

11. Replication of the E. coli Chromosome is Bidirectional

12. Replication of the E. coli Chromosome is Semidiscontinuous Replicates continuously DNA synthesis is going in same direction as replication fork Replicates discontinuously DNA synthesis is going in opposite direction as replication fork Joined by DNA ligase Because of the anti-parallel structure of the DNA duplex, new DNA must be synthesized in the direction of fork movement in both the 5’ to 3’ and 3’ to 5’ directions overall. However all known DNA polymerases synthesize DNA in the 5’ to 3’ direction only. The solution is semidiscontinuous DNA replication.

13. “Now this end is called the thagomizer, after the late Thag Simmons.”

14. Klenow Fragment DNA Repair (Errors fixed after DNA replication) No Proofreading

16. Nick Translation 5’  3’ exonuclease activity digests DNA 5’  3’ polymerase activity replaces the digested DNA with new DNA They act together to edit out sections of damaged DNA

20. Main replicative enzyme Repair enzyme

21. Okazaki fragment RNA Roles of DNA Pol III and Pol I in E. coli Pol III—main DNA replication enzyme. It exists as a dimer to coordinate the synthesis of both the leading and lagging strands at the replication fork. Pol I—repair enzyme to remove RNA primers that initiate DNA synthesis on both strands. It is need predominantly for maturation of Okazaki fragments. 1) Removes RNA primers (5’3’ Exo) 2) Replaces the RNA primers with DNA (5’3’ Pol & 3’5’ Exo proofreading) >10 kb Q: Why do Okazaki fragments initiate with RNA primers? A: Because DNA polymerases require a primer but can’t synthesize them de novo DNA Pol I 1 kb