1. Prokaryotic DNA Replication
Nov. 12, 2004
MBB 407/511
Lecture 19:
Prokaryotic DNA Replication
(Part I)

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

3. I. Why Study DNA Replication?
1) To understand cancer
2) To understand aging
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)

4. II. Historical Background
A Watson and Crick: DNA Structure Predicts a Mechanism of Replication
“It has no escaped our notice that the specific pair we have postulated immediately suggests
a possible copying mechanism for the genetic material.”
B Meselson and Stahl: DNA Replication is Conservative

5. The Meselson-Stahl Experiment
“the most beautiful experiment in biology.”
All
hybrids
1/2 old:
1/2 new
1/2 hybids:
1/2 new
1/4 old:
3/4 new
All
hybrids
Three potential DNA replication models and their predicted outcomes
The actual data!

6. III. General Features of DNA Replication
DNA Synthesis:
1. requires a DNA template and a primer with a 3’ OH end.
(DNA synthesis cannot initiate de novo)
Short RNA molecules
act as primersin vivo
2. requires dNTPs.
3. occurs in a 5’ to 3’ direction.

7. Replication of the E. coli Chromosome is Bidirectional

8. 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.

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

11. Model for the Interaction of Klenow Fragment with DNA

12. How the Proofreading Activity of Klenow Fragment Works

13. DNA Polymerase I can Perform “Nick Translation”
They act together to edit out
sections of damaged DNA

18. 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
DNA Pol I
1 kb
RNA
Okazaki fragment
RNA primer replaced with
DNA by Pol I’s nick translatiton activity
Okazaki fragment