1. Authored by Peter J. Russell
CHAPTER 3 DNA Replication

2. Semiconservative DNA Replication
The Watson and Crick DNA model implies a mechanism for replication (very important!):
DNA can be denatured and separated
A complementary copy can be produced for each strand
3 models were proposed for DNA replication:
The conservative model proposed that both strands of one copy would be entirely old DNA, while the other copy would have both strands of new DNA
The dispersive model proposed that dsDNA would fragment (dangerous idea!), replicate and reassemble.
The semiconservative model proposed that DNA strands separate and a complementary strand is synthesized for each
in other words (or pictures)…

3. 3 Models proposed for DNA replication
Figure 3.1 Three models for DNA replication. (a) Semiconservative model (the correct model). (b) Conservative model. (c) Dispersive model. The parental strands are shown in taupe, and the newly synthesized strands are shown in red.

4. Meselson-Stahl Experiment
Meselson and Stahl grew E. coli in a heavy, non- radioactive isotope of nitrogen. (15N in the form of 15NH4Cl salt). Because it is heavier, DNA containing 15N is more dense than DNA with normal 14N and can be separated by cesium chloride (CsCl) density gradient centrifugation

5. Figure 3.1 Schematic diagram for separating DNAs of different buoyant densities by equilibrium centrifugation in a cesium chloride density gradient. The separation of 14N-DNA and 15N-DNA is illustrated.

6. Animation: Meselson-Stahl Experiment
After the E. coli were incubated with heavy 15N, the investigators shifted the cells to a medium containing normal 14N, and took cell samples at different time points
DNA was then extracted from the treated cells and each sample was subjected to cesium chloride density centrifugation for content analysis
Animation: Meselson-Stahl Experiment

7. Figure 3. 2 The Meselson–Stahl experiment
Figure 3.2 The Meselson–Stahl experiment. The demonstration of semiconservative replication in E. coli. Cells were grown in a 15N-containing medium for several replication cycles and then were transferred to a 14N-containing medium. At various times over several replication cycles, samples were taken; the DNA was extracted and analyzed by CsCl equilibrium density gradient centrifugation. Shown in the figure are a schematic interpretation of the DNA composition after various replication cycles, photographs of the DNA bands, and densitometric scans of the bands.

8. Subsequent work by other groups demonstrated that eukaryotes also replicate their DNA semiconservatively
Replicating chinese hamster ovary (CHO) cells were incubated in media with bromodeoxyuridine (BUdR), a non-reutilized precursor of DNA that is detected by monoclonal antibody
Harlequin chromosomes ware produced: sister chromatids that stain differently, so that one appears dark and the other light (harlequin-like)
Figure 3.3 Visualization of semiconservative DNA replication in eukaryotes. Shown are harlequin chromosomes in Chinese hamster ovary cells that have been allowed to go through two rounds of DNA replication in the presence of the base analog 5-bromodeoxyuridine, followed by staining. Arrows indicate the sites where crossing-over has occurred.

9. DNA Polymerases
Arthur Kornberg isolated the Kornberg enzyme; it was renamed DNA polymerase (thankfully!). His son proved to be a successful biologist as well…
In vitro DNA synthesis required 5 components:
All four deoxynucleotide tri-phosphates; dATP, dTTP, dCTP and dGTP (substrates)
DNA polymerase I (enzyme)
DNA (template matter)
Primer DNA (is actually RNA in vivo)
Magnesium ions (divalent cations either alter the conformation of the enzyme or substrate, or take part in the chemistry of the catalytic reaction at the active site by stabilizing the intermediate species)

10. Animation: DNA synthesis
Characteristics of
DNA polymerase
joins the deoxynucleotides to generate a polynucleotide
joining takes place between the 5’ phosphate of the new nucleotide and the 3’ OH of the growing DNA strand
energy for the bonding comes from the catabolism of an internal phosphate bond (use 2 equivalents of ATP).
The incoming nucleotide is selected based on the complementary base encoded on the template
DNA polymerase can only elongate the strand in the 5’  3’ direction
Animation: DNA synthesis

11. Prokaryotic DNA Polymerase Family
DNA pol I
Replicates DNA in the 5’  3’ direction
Composed of a single peptide
3’ to 5’ exonuclease activity (proofreading)
5’ to 3’ exonuclease activity (primer removal)
DNA Pol II, IV, V
Single peptide enzyme
DNA repair functions
DNA Pol III
Catalytic core is composed of 3 peptide subunits (a, e, q)
Complete holoenzyme is composed of an additional 6 peptide subunits (making the total 9 peptides)
5’ to 3’ exonuclease activity
3’ to 5’ exonuclease activity
No repair function is observed

12. Molecular Model of DNA Replication
The copying of DNA is remarkable in its speed and accuracy, using a dozen enzymes and other proteins
Replication begins at special DNA sequences called replicators within origins of replication, where the two DNA strands are separated by origin of replication enzymes, opening a replication “bubble” with 2 “forks”
A eukaryotic chromosome has thousands of origins of replication; prokaryotes have one origin (called OriC)
Replication proceeds in both directions from each origin, until the entire molecule is copied
Animation: Origins of Replication

13. Many Replication bubbles
Origin of replication (OriC)
Parental (template) strand
Daughter (new) strand
Double-
stranded
DNA molecule
1 Replication bubble
Replication forks
0.5 µm
Two daughter DNA molecules
(a) Origin of replication in E. coli
Origin of replication
Double-stranded DNA molecule
Parental (template) strand
Daughter (new) strand
0.25 µm
Replication forks
Many Replication bubbles
Two daughter DNA molecules
(b) Origins of replication in eukaryotes

14. Players involved in DNA Replication
Origin of Replication Enzymes (Initiator Proteins) recognize the start site on template DNA and initially open up the DNA (single strands)
Helicases untwist the double helix at the replication forks
Primase begins DNA replication by laying an an RNA primer (10 nt long) for DNA polymerase to start at
Single-strand binding proteins bind to and stabilize the single-stranded DNA until it can be used as a template
DNA polymerase polymerizes new DNA in a 5’ to 3’ direction, proofreads the newly laden DNA, excises error ridden DNA and replaces that DNA with the correct sequence. Speed/fidelity: mutates 1 X 10-10
Topoisomerase corrects “overwinding” ahead of replication forks by breaking, swiveling, and rejoining DNA strands
Terminal replication proteins stall replication fork progress for polymeriation to catch-up
DNA ligase anneals nucleotides together, creating a DNA strand

15. Redundant slide showing the function of the proteins and their associated gene names

16. Initiation of Replication
Replication is bidirectional from a specific sequence of DNA called the origin of replication (OriC)
DnaA (Initiator protein) binds to the 9 bp repeat sequence within OriC, causing local denaturation of the helix within the 13 bp repeat
DNA helicase is recruited (by DnaA) and loaded (by DnaC), and untwists the DNA helix (use ATP)
Helicase also recruits DNA primase (modified RNA pol), forming the primosome complex, which makes an RNA primer of nucleotides) for DNA synthesis
Helical tension is relieved by DNA gyrase (topoisomerase)
Figure 3.5 Initiation of replication in E. coli. The DnaA initiator protein binds to oriC (the replicator) and stimulates denaturation of the DNA. DNA helicases are recruited and begin to untwist the DNA to form two head-to-head replication forks.

17. Semidiscontinuous DNA Replication
The antiparallel structure of the double helix, with two strands oriented in opposite directions, introduces an interesting paradox to replication
DNA polymerases add nucleotides only to the free 3end of a growing strand; therefore, a new DNA strand can elongate only in the 5 to 3direction
Along one template strand of DNA, DNA polymerase synthesizes a leading strand continuously, moving toward the replication fork
To elongate the other new strand, called the lagging strand, DNA polymerase must work in the direction away from the replication fork
The lagging strand is synthesized as a series of segments called Okazaki fragments, which are joined together by DNA ligase (semidiscontinuous process) (cool experiment! – see book)

18. Animation: DNA synthesis
Replisome model: the complex of key replication proteins with the DNA at the replication fork
Animation: DNA synthesis

19. Replication
Summary
Figure 3.6 Model for the events occurring around a single replication fork of the E. coli chromosome. (a) Initiation. (b) Further untwisting and elongation of the new DNA strands. (c) Further untwisting and continued DNA synthesis. (d) Removal of the primer by DNA polymerase I. (e) Joining of adjacent DNA fragments by the action of DNA ligase. Green=RNA; red=new DNA.

20. Ligation
Summary

21. Strand
Synthesis
Summary

22. Rolling Circle Replication
Observed in some viruses (lambda)
Begins with a nick (single-stranded break) at the origin (O).
The 5’ end is displaced from the strand and the 3’ end acts as a primer for DNA polymerase III, which synthesizes a continuous strand using the intact DNA molecule as a template
The 5’ end continues to be displaced as the circle “rolls” and is protected by SSB proteins until discontinuous DNA synthesis makes it a ds DNA again. But hold on, we just made circular and linear DNA!
Figure 3.10 The replication process of double-stranded circular DNA molecules through the rolling circle mechanism. The active force that unwinds the 5’ tail is the movement of the replisome propelled by its helicase components.

23. Chromosome structure and phage life-cycle
Linear is packaged; Circular is replicated…

24. DNA Replication in Eukaryotes
Prokaryotic and eukaryotic DNA replication is similar but for the size, linearity, rate and number of eukaryotic chromosomes
Eukaryotes have multiple origins of replication. The DNA replicated from a single origin is called a replicon
Replicon size is smaller in eukaryotes. Replication is slower and each chromosome contains many replicons which are activated at distict times
Each replicon is used only once when DNA is replicated in the S phase of cell cycle. How you ask? Good question…

25. Keeping DNA replication in eukaryotes real…
Replicators are less well defined in higher eukaryotes
Yeast have Autonomously Replicating Sequences (ARSs)
In eukaryotes, the key issue is not to use any origin more than once (this would be absolutely disastrous)! To avoid this, these steps are followed:
First, replicator selection occurs. The multisubunit initiator protein, Origin Recognition Complex (ORC), binds to the replicator during the G1 phase of the cell cycle
Next, other initiator proteins (including licencing factors, which are produced only during G1) assemble on the replicator to form pre-replicative complexes (pre-RC).
When the cell enters the S phase, specific cyclin dependent kinases (Cdks) are present, causing a release and deactivation of licencing factors, allowing the DNA helix to unwind, thus initiating replication
Importantly, the presence of these specific Cdks inhibits another round of pre-RC formation until the cell reenters G1 after a completed round of DNA replication

26. Eukaryotic DNA polymerase (blah, blah…)
15 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 e (epsilon) extend the RNA/DNA primers, one on the leading strand and the other on the lagging strand (it is not clear which synthesizes which)
Other DNA pols replicate mitochondrial or chloroplast DNA or 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

27. Replicating Chromosome Ends5
Ends of parental DNA strands
Leading strand
Replicating Chromosome Ends
Lagging strand 3
Limitations of DNA polymerase create problems for the linear DNA of eukaryotic chromosomes (remember that bacteria and viruses replicate circular DNA and do not suffer from this dilemma)
The usual replication machinery provides no way to complete the terminal 5 end of a DNA strand
As DNA polymerase has no primer to start off at, repeated rounds of replication produce shorter and shorter progeny DNA strands, effecting the cell
Last fragment
Previous fragment
RNA primer
Lagging strand 5 3
Parental strand
Removal of primers and replacement with DNA where a 3 end is available 5 3
Second round of replication
Figure Shortening of the ends of linear DNA molecules 5
New leading strand 3
New lagging strand 5 3
Further rounds of replication
Shorter and shorter daughter molecules

28. the solution…
It has been proposed the shortening of telomeres contributes to aging
Dilemma: If chromosomes of germ cells became shorter in every cell cycle, the essential genes would eventually be missing from the gametes they produce, destroying the heritability of the species
Eukaryotic chromosomal DNA molecules have terminal nucleotide sequences called telomeres (5’ TTAGGG 3’). Telomeres do not prevent shortening of DNA; they postpone genetic erosion near the ends of DNA
Solution: An enzyme called telomerase catalyzes the lengthening of telomeres in germ cells (sperm and egg cells), thus protecting the integrity of reproduction
Of note: The shortening of telomeres protects somatic cells from cancerous growth by limiting the number of cell divisions (and hence compiled mutations). There is evidence of telomerase activity in cancer cells, allowing cancer cells to persist

29. The alternative strand of DNA gets filled in using DNA as a template
Elizabeth Blackburn and Carol Greider have shown that chromosome lengths are maintained by telomerase
The enzyme is composed of protein and RNA. The RNA component (451 bases in humans) includes an 11 base template RNA sequence that is used for the synthesis of new telomere repeat DNA. Thus, telomerase acts as a reverse transcriptase (TERT)
The 3’CAAUC5’ sequence on RNA interacts with the 5’GTTAG3’ sequence on DNA. The remaining 3’CCAAUC5’ sequence on RNA acts as a template to fill in the 5’GGTTAG3’ sequence on DNA. The process repeats and telomerase drops off
The alternative strand of DNA gets filled in using DNA as a template
Figure 3.15 Synthesis of telomeric DNA by telomerase. The example is of Tetrahymena telomeres. The process is described in the text. (a) The starting point is the chromosome end with5’ gap left after primer removal. (b) Binding of telomerase to the overhanging telomere repeat at the end of the chromosome. (c) Synthesis of three-nucleotide DNA segment at chromosome end, using the RNA template of telomerase. (d) The telomerase moves so that the RNA template can bind to the newly synthesized TTG in a different way. (e) Telomerase catalyzes the synthesis of a new telomere repeat, using the RNA template. The process recurs, to add more telomere repeats. (f) After telomerase has left, new DNA is made on the template, starting with an RNA primer. (g) After the primer is removed, the result is a longer chromosome than at the start, with a new 5’ gap.

30. Assembling Newly Replicated DNA into Nucleosomes
When eukaryotic DNA is replicated, it complexes with histones. This requires the synthesis of histone proteins and the assembly of new nucleosomes.
Transcription of histone genes is initiated near the end of the G1 phase and the translation of histone proteins occurs throughout S phase
An H3/H4 tetramer is reused in 1 new strand
H2A/H2B is broken down to 2 dimers which are reused arbitrarily

32. III
III I I
DNA

33. Replication of Circular DNA and the Supercoiling Problem
Some circular chromosomes (e.g., E. coli) are circular throughout replication, creating a theta-like q shape.
As the strands separate on one side of the circle, positive supercoils form elsewhere in the molecule.
Topoisomerases relieve the supercoils, allowing the DNA strands to continue separating as the replication forks advance.
Figure 3.9 Bidirectional replication of circular DNA molecules.