1. DNA Replication in Prokaryotes and Eukaryotes
Overall mechanism
Roles of Polymerases & other proteins
More mechanism: Initiation and Termination
Mitochondrial DNA replication

2. DNA replication is semi-conservative, i. e
DNA replication is semi-conservative, i.e., each daughter duplex molecule contains one new strand and one old.

3. Does DNA replication begin at the same site in every replication cycle
Does DNA replication begin at the same site in every replication cycle? Electron microscope image of an E. coli chromosome being replicated. Structure (theta, θ) suggests replication started in only one place on this chromosome.
Fig 20.9 in 4th edition.
Fig. 20.9

4. Does DNA replication begin at the same site in every replication cycle?
Experiment:
Pulse-label a synchronized cell population during successive rounds of DNA replication with two different isotopes, one that changes the density of newly synthesized DNA (15N), and one that makes it radioactive (32P).
DNA is then isolated, sheared, and separated by CsCl density gradient ultra-centrifugation.
Radioactivity (32P) in the DNAs of different densities is counted.

7. Prior to 1st replication cycle, 15N (which incorporates into the
Prior to 1st replication cycle, 15N (which incorporates into the bases of DNA) was added for a brief period
Prior to 2nd replication cycle, cells were pulsed with 32P (which gets incorporated into the phosphates of replicating DNA)
15N - heavy isotope of Nitrogen
32P - radioactive isotope of phosphorus
1st

12. DNA is isolated, sheared into fragments, and separated by CsCl-density gradient centrifugation.

14. Same Origin Random Origins
Blow up of the last 2 rows of DNA in the previous slide (i.e., labeled DNA, and labeled, sheared DNA).
Same Origin
Random Origins
Labeled, sheared DNA
Labeled DNA

15. Result:
~50% (the most possible) of the incorporated 32P was in the same DNA that was shifted by 15N
Conclusion:
Replication of bacterial chromosome starts at the same place every time
Conclusion

16. Using Electron Microscopy (EM) to Demonstrate that DNA Replication is Bi-Directional
Pulse-label with radioactive precursor (3H-thymidine)
Then do EM and autoradiography.
Has been done with prokaryotes and eukaryotes.

17. Conclusion: eukaryotic origins also replicate bi-directionally!
Drosophila cells were labeled with a pulse of highly radioactive precursor, followed by a pulse of lower radioactive precursor; then replication bubbles were viewed by EM and autoradiography.
Fig a in 4th edition - Drosophila
Conclusion: eukaryotic origins also replicate bi-directionally!
Fig in Weaver

18. Another way to see that DNA replication is
Bi-directional --
Cleave replicating
SV40 viral DNA with a
restriction enzyme that
cuts it once.
SV40 virus replicating in animal cell . Similar to Fig in Weaver
Similar to Fig in Weaver 4

19. Replicon - DNA replicated from a single origin
Table in notes
Eukaryotes have many replication origins.

20. Enzymology of DNA replication: implications for mechanism
1. DNA-dependent DNA polymerases
synthesize DNA from dNTPs
require a template strand and a primer strand with a 3’-OH end
all synthesize from 5’ to 3’ (add nt to 3’ end only)

21. Movie – DNA polymerization
P-P : pyrophosphate, cleaved by pyrophosphatase, recycle the P
Note: what happens to the P-P?

22. Comparison of E.coli DNA Polymerases I and III
1 subunit
10 subunits
Table in notes

23. Proofreading Activity
Insertion of the wrong nucleotide causes the DNA polymerase to stall, and then the 3’-to-5’ exonuclease activity removes the mispaired A nt. The polymerase then continues adding nts to the primer.
Fig in Weaver 4

24. If DNA polymerases only synthesize 5’ to 3’, how does the replication fork move directionally?

25. Lagging strand synthesized as small (~100-1000 bp) fragments - “Okazaki fragments” .
Okazaki fragments begin as very short 6-15 nt RNA primers synthesized by primase.
2. Primase - RNA polymerase that synthesizes the RNA primers (11-12 nt that start with pppAG) for both lagging and leading strand synthesis

26. Lagging strand synthesis (continued)
Pol III extends the RNA primers until the 3’ end of an Okazaki fragment reaches the 5’ end of a downstream Okazaki fragment.
Then, Pol I degrades the RNA part with its 5’-3’ exonuclease activity, and replaces it with DNA. Pol I is not highly processive, so stops before going far.

27. At this stage, Lagging strand is a series of DNA fragments (without gaps).
Fragments stitched together covalently by DNA Ligase.
3. DNA Ligase - joins the 5’ phosphate of one DNA molecule to the 3’ OH of another, using energy in the form of NAD (prokaryotes) or ATP (eukaryotes). It prefers substrates that are double-stranded, with only one strand needing ligation, and lacking gaps.

28. DNA Ligase Substrate Specificity
Lower efficiency for some, not “not”

29. + + Mechanism of Prokaryotic DNA Ligase
Ligase cleaves NAD and attaches to AMP.
Ligase-AMP binds and attaches to 5’ end of DNA #1 via the AMP.
The 3’OH of DNA #2 reacts with the phosphodiester shown, displacing the AMP-ligase.
AMP & ligase separate. HO P 3' 5'
Ligase P
NAD
NMN
+AMP 1 N M N 3' P
Ligase 1 + P
AMP
AMP 3' N A D 2 HO
Also in notes +
AMP 1 2 P 3' 5'
(Euk. DNA ligase uses ATP as AMP donor)

30. Movie - Bidirectional Replication: Leading and lagging strand synthesis

31. Other proteins needed for DNA replication:
4. DNA Helicase (dnaB gene) – hexameric protein, unwinds DNA strands, uses ATP.
5. SSB – single-strand DNA binding protein, prevents strands from re-annealing and from being degraded, stimulates DNA Pol III.
6. Gyrase – a.k.a. Topoisomerase II, keeps DNA ahead of fork from over winding (i.e., relieves torsional strain).
Replisome - DNA and protein machinery at a replication fork.

32. DNA Helicase (dnaB gene) Assay
The two bands in the substrate lane (Sub) are probably from linear and circular forms of the large molecule to the left. If true, then the linear is probably the bottom one.
Fig in Weaver

33. Helicase – the movie

34. Replication Causes DNA to Supercoil

35. Rubber Band Model of Supercoiling DNA
DNA Gyrase relaxes positive supercoils by breaking and rejoining both DNA strands.
Figure not in the 4th Edition.