Base Pairing
The rules of base pairing (or nucleotidepairing) are:
- A with T: the purine adenine (A) always pairs with the pyrimidine thymine (T)
- C with G: the pyrimidine cytosine (C) always pairs with the purine guanine (G)
This is consistent with there not being enough space (20 Å) for two purines to fit within the helix and too much space for two pyrimidines to get close enough to each other to form hydrogen bonds between them.
But why not A with C and G with T?
The answer: only with A & T and with C & G are there opportunities to establish hydrogen bonds (shown here as dotted lines) between them (two between A & T; three between C & G). These relationships are often called the rules of Watson-Crick base pairing, named after the two scientists who discovered their structural basis.
The rules of base pairing tell us that if we can “read” the sequence of nucleotides on one strand of DNA, we can immediately deduce the complementary sequence on the other strand.
The rules of base pairing explain the phenomenon that whatever the amount of adenine (A) in the DNA of an organism, the amount of thymine (T) is the same (called Chargaff’s rule). Similarly, whatever the amount of guanine (G), the amount of cytosine (C) is the same.
| Relative Proportions (%) of Bases in DNA | ||||
|---|---|---|---|---|
| Organism | A | T | G | C |
| Human | 30.9 | 29.4 | 19.9 | 19.8 |
| Chicken | 28.8 | 29.2 | 20.5 | 21.5 |
| Grasshopper | 29.3 | 29.3 | 20.5 | 20.7 |
| Sea Urchin | 32.8 | 32.1 | 17.7 | 17.3 |
| Wheat | 27.3 | 27.1 | 22.7 | 22.8 |
| Yeast | 31.3 | 32.9 | 18.7 | 17.1 |
| E. coli | 24.7 | 23.6 | 26.0 | 25.7 |
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24 May 2006
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DNA Replication
Before a cell can divide, it must duplicate all its DNA. In eukaryotes, this occurs during S phase of the cell cycle.
The Biochemical Reactions
- DNA replication begins with the “unzipping” of the parent molecule as the hydrogen bonds between the base pairs are broken.
- Once exposed, the sequence of bases on each of the separated strands serves as a template to guide the insertion of a complementary set of bases on the strand being synthesized.
- The new strands are assembled from deoxynucleoside triphosphates.
- Each incoming nucleotide is covalently linked to the “free” 3′ carbon atom on the pentose (figure) as
- the second and third phosphates are removed together as a molecule of pyrophosphate (PPi).
- The nucleotides are assembled in the order that complements the order of bases on the strand serving as the template.
- Thus each C on the template guides the insertion of a G on the new strand, each G a C, and so on.
- When the process is complete, two DNA molecules have been formed identical to each other and to the parent molecule.
The Enzymes
- A portion of the double helix is unwound by a helicase.
- A molecule of a DNA polymerase binds to one strand of the DNA and begins moving along it in the 3′ to 5′ direction, using it as a template for assembling a leading strand of nucleotides and reforming a double helix. In eukaryotes, this molecule is called DNA polymerase epsilon (ε).
- Because DNA synthesis can only occur 5′ to 3′, a molecule of a second type of DNA polymerase (delta, δ, in eukaryotes) binds to the other template strand as the double helix opens. This molecule must synthesize discontinuous segments of polynucleotides (called Okazaki fragments). Another enzyme, DNA ligase I then stitches these together into the lagging strand.
| External Link |
| Link to John Kyrk’s excellent animation of the entire process. |
| Please let me know by e-mail if you find a broken link in my pages.) |
DNA Replication is Semiconservative
When the replication process is complete, two DNA molecules — identical to each other and identical to the original — have been produced. Each strand of the original molecule has
- remained intact as it served as the template for the synthesis of
- a complementary strand.
This mode of replication is described as semi-conservative: one-half of each new molecule of DNA is old; one-half new.
Watson and Crick had suggested that this was the way the DNA would turn out to be replicated. Proof of the model came from the experiments of Meselson and Stahl. [Link to them.]
Speed of Replication
Bacteria
The single molecule of DNA that is the E. coli genome contains 4.7 x 106 nucleotide pairs. DNA replication begins at a single, fixed location in this molecule, the replication origin, proceeds at about 1000 nucleotides per second, and thus is done in no more than 40 minutes. And thanks to the precision of the process (which includes a “proof-reading” function), the job is done with only about one incorrect nucleotide for every 109 nucleotides inserted. In other words, more often than not, the E. coli genome (4.7 x 106) is copied without error!
Eukaryotes
The average human chromosome contains 150 x 106 nucleotide pairs which are copied at about 50 base pairs per second. The process would take a month (rather than the hour it actually does) but for the fact that there are many places on the eukaryotic chromosome where replication can begin. Replication begins at some replication origins earlier in S phase than at others, but the process is completed for all by the end of S phase. As replication nears completion, “bubbles” of newly replicated DNA meet and fuse, finally forming two new molecules.
Control of Replication
With their multiple origins, how does the eukaryotic cell know which origins have been already replicated and which still await replication?
An observation: When a cell in G2 of the cell cycle is fused with a cell in S phase, the DNA of the G2 nucleus does not begin replicating again even though replication is proceeding normally in the S-phase nucleus. Not until mitosis is completed, can freshly-synthesized DNA be replicated again.
Two control mechanisms have been identified — one positive and one negative. This redundancy probably reflects the crucial importance of precise replication to the integrity of the genome.
Licensing: positive control of replication
In order to be replicated, each origin of replication must be bound by:
- an Origin Recognition Complex of proteins (ORC). These remain on the DNA throughout the process.
- Accessory proteins called licensing factors. These accumulate in the nucleus during G1of the cell cycle. They include:
- Cdc-6 and Cdt-1, which bind to the ORC and are essential for coating the DNA with
- MCM proteins. Only DNA coated with MCM proteins (there are 6 of them) can be replicated.
Once replication begins in S phase,
- Cdc-6 and Cdt-1 leave the ORCs (the latter by ubiquination and destruction in proteasomes).
- The MCM proteins leave in front of the advancing replication fork.
Geminin: negative control of replication
G2 nuclei also contain at least one protein — called geminin — that prevents assembly of MCM proteins on freshly-synthesized DNA (probably by blocking the actions of Cdt1).
As the cell completes mitosis, geminin is degraded so the DNA of the two daughter cells will be able to respond to licensing factors and be able to replicate their DNA at the next S phase.
| Some cells deliberately cut the cell cycle short allowing repeated S phases without completing mitosis and/or cytokinesis. This is called endoreplication. How these cells regulate the factors that normally prevent DNA replication if mitosis has not occurred is still being studied. Endoreplication is described on a separate page. Link to it. |
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25 March 2012
The Meselson – Stahl Experiment
“the most beautiful experiment in biology”
DNA Replication is Semiconservative
The structure of DNA suggested to Watson and Crickthe mechanism by which DNA — hence genes — could be copied faithfully. They proposed that when the time came for DNA to be replicated, the two strands of the molecule
- separated from each other but
- remained intact as each served as the template for the synthesis of
- a complementary strand.
When the replication process is complete, two DNA molecules — identical to each other and identical to the original — have been produced.
This mode of replication is described as semiconservative: one-half of each new molecule of DNA is old; one-half new.
Link to discussions of:
|
While Watson and Crick had suggested that this was the way the DNA would turn out to be replicated, proof of the model came from the experiments of M. S. Meselson and F. W. Stahl.
They grew E. coli is a medium using ammonium ions (NH4+) as the source of nitrogen for DNA (as well as protein) synthesis. 14N is the common isotope of nitrogen, but they could also use ammonium ions that were enriched for a rare heavy isotope of nitrogen, 15N.
After growing E. coli for several generations in a medium containing 15NH4+, they found that the DNA of the cells was heavier than normal because of the 15N atoms in it.
The difference could be detected by extracting DNA from the E. coli cells and spinning it in an ultracentrifuge. The density of the DNA determines where it accumulates in the tube.
Then they transferred more living cells that had been growing in 15NH4+ to a medium containing ordinary ammonium ions (14NH4+) and allowed them to divide just once.
The DNA in this new generation of cells was exactly intermediate in density between that of the previous generation and the normal.
This, in itself, is not surprising. It tells us no more than that half the nitrogen atoms in the new DNA are 14N and half are 15N. It tells us nothing about their arrangement in the molecules.
However, when the bacteria were allowed to divide again in normal ammonium ions (14NH4+), two distinct densities of DNA were formed:
- half the DNA was normal and
- half was intermediate.
As this interpretative figure indicates, their results show that DNA molecules are not degraded and reformed from free nucleotides between cell divisions, but instead, each original strand remains intact as it builds a complementary strand from the nucleotides available to it.
This is called semiconservative replication because each daughter DNA molecule is one-half “old” and one-half “new”.
Immortal strands. Note that the “old” strand (the red one in the top half of the figure) is immortal because — barring mutations or genetic recombination — it will continue to serve as an unchanging template down through the generations.
E. coli is a bacterium, but semiconservative replication of DNA also occurs in eukaryotes. And because each DNA molecule in a eukaryote is incorporated in one chromosome, the replication of entire chromosomes is semiconservative as well. This also means that the eukaryotic chromosome contains one “immortal strand” of DNA.
| Link to an illustrated demonstration of the semiconservative replication of chromosomes and the significance of their “immortal strands”. |
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28 January 2012
dodi ' s blog biology 1 
