Biology 161 Molecular Basis of Inheritance: DNA Structure and Replication

Fall 2000

A. Experiments that convinced people that DNA is the genetic material

Early ideas were that the genetic material might be either protein or nucleic acid.
Chromosomes in eucaryotic cells are constructed of both protein and DNA.

A series of experiments, done by different people and spanning a time period of nearly 25 years, began with the experiment we talked about in the laboratory several weeks ago, the experiment with streptococcal bacteria and mice done by Griffiths in 1928.

Griffiths showed that something (he called it the transforming principle) was released by dead, virulent, smooth-coated bacterial cells. The substance appeared to be taken up by live, non-virulent, rough-coated bacterial cells of the same species and converted them (transformed them) to virulent, smooth-coated cells.

During the next decade or so, Avery and his colleagues at Rockefeller University in New York City, worked on the problem of trying to identify what the transforming principle was. Others had identified enzymes such as proteases, ribonucleases (RNAses), and deoxyribonucleases (DNAses). When the transforming principle was treated with DNAse, it failed to transform the rough bacteria. None of the other enzymes had such an effect. The conclusion: that DNA was the transforming principle.

Other scientists were not convinced that this was right.

In the early 1950’s, another team (Alfred Hershey and Martha Chase) did an experiment that was more convincing. By this time people knew that some viruses (e. g., T2 bacteriophage) could infect E. coli bacteria.

T2 virus consisted only of DNA and proteins.
The virus bodies seemed to remain on the outside of the infected cells: people had seen this in electron micrographs of infected cells at different stages.
However, the viral genetic material directed the bacterial cells to make lots of new viruses.
Also, radioisotopes were available to use for studying cells and viruses.
Radioactive sulfur could be used to specifically label proteins.
Radioactive phosphorus could be used to specifically label DNA and RNA.

Hershey and Chase used these two isotopes to separately label two batches of viruses.

They then infected two batches of bacterial cells in their nutrient medium (or soup).
After a short time, they mixed the cells vigorously in a blendor, a treatment that broke away any viruses that were still attached to the outsides of the bacteria.
They centrifuged the stuff to separate the bacteria from the soup containing the removed viruses.

Analysis showed

the virus bodies on the outsides of the bacteria contained proteins but no DNA
the viral DNA was all on the inside of the bacteria, but no viral proteins.
If they let the viral infection of the bacteria continue even after they removed the viral stuff on the outsides of the cell, new viruses were produced and burst out of the bacterial cells.

Conclusion: the viral DNA had directed the entire production of new viruses; therefore, it was the genetic material for the virus.

Other evidence for considering DNA as the genetic material:

Diploid cells had twice as much DNA as haploid cells.
DNA composition varies from one kind of organism to another.
The amount of the 4 nucleotides are not present in equal amounts, but the mole % of A always equals the mole % of T, and the mole % of G always equals the mole % of C (Chargaff’s rules).

B. The Double Helix

In the 1950’s, after people knew what the structure of a nucleotide is, the most important research about DNA seemed to be the quest to figure out its three-dimensional structure. (See story in the text about the work done by Wilkins, Franklin, Watson and Crick. Wilkins and Franklin did the extensive X-ray diffraction work and were considering how the structure was organized. Watson and Crick figured out the structure by building wire and stick models based on the data that had been accumulated by Wilkins and Franklin. The Watson and Crick paper describing the structure was published in 1953.)

X-ray diffraction techniques result in a picture of dots and spaces that represent the positions of atoms in the crystals of a molecule.
There are mathematical rules, based on chemistry, for figuring out where the atoms actually lie and how they are attached to each other.
The DNA molecule had dimensions that were important in solving this puzzle: a constant width of about 2 nm (= 2 strands of material) and a helical arrangement with turns at particular distances (3.4 nm). (The length varies for each chromosome or piece of DNA.)

 

Remember that the amounts of A and T bases are equal to each other; the amounts of G and C are equal to each other.

 

A and G are purines (double rings); T and C are pyrimidines (single rings).

If the bases lie on the inside of the double strands and are paired A-T and G-C, they fit and they make hydrogen bonds in the right places.

The base pairing rules say how the strands will be opposite to each other, but these rules do not specify the line-up (i. e., the linear sequence) of the base pairs. The linear sequences of different genes are different; the sequences of the same gene from one species to another is usually at least somewhat different.

The explanation of DNA structure allowed accurate prediction about the mechanism of DNA replication.

C. Pattern of DNA replication.

The essential pattern of DNA replication is semi-conservative.

Each DNA molecule consists of two strands that are complementary to each other, A always opposite T and G always opposite C.
The two strands are separated from each other (disconnection of the hydrogen bonds).
Each nucleotide of each "old" strand attracts its complementary nucleotide according to the base pairing rule.
After the new nucleotides settle into place opposite the old ones, they are linked sugar-to-phosphate by DNA polymerase enzymes.
This happens one base pair at a time along each DNA strand.

Please note: the nucleotides are not attracted into place or put into place by the DNA polymerase. The polymerase only attaches them to each other to form the new strand after they have been attracted to their complementary partners. The hydrogen bonds between the bases form without enzymatic help.

The semi-conservative mode of replication was demonstrated experimentally by Meselson and Stahl.

Heavy nitrogen atoms (an isotope of nitrogen) were available for labelling DNA.
Bacteria were grown for a while in a medium containing heavy nitrogen to allow them to make DNA containing this isotope.
They were then transferred to a medium containing the more abundant light form of nitrogen so that they could spend a short time replicating DNA without the label.
The cells were broken apart, and the DNA molecules purified out. They were centrifuged in an ultra high speed centrifuge to separate any heavy DNA, light DNA, and heavy/light DNA molecules that might be present.

The logic behind the experiment:

If DNA were copied, but the old DNA retained its double stranded structure and all new DNA were new double strands, then two bands of DNA would be present after centrifugation, heavy and light (conservative replication).
If DNA were copied so that the resulting DNA molecules each contained one heavy strand and one light strand, then the heavy/light molecules would form a band that would settle midway between the positions of all heavy and all light DNA (semi-conservative replication).
If the DNA were copied so that the resulting DNA molecules each contained some heavy stuff and some light stuff, there would also be the heavy/light band after centrifugation (dispersive replication).

The result: a heavy/light band. Could be semi-conservative or dispersive, but not conservative.

Now, how to tell if the process is conservative or dispersive:

Let replication go on longer so that the first set of replicated DNA strands are allowed to replicate again.
If replication is conservative, light strands in the heavy/light molecules should serve as templates for more light strands. The resulting DNA molecules should include both all light molecules and heavy/light hybrids. (This is what resulted.)
If replication is dispersive, the next round of replication should still only yield heavy/light combination DNA. (This did not happen.)

D. DNA double strands are anti-parallel.

We say that the DNA molecules consist of anti-parallel strands because the orientation of the sugars and phosphates of the nucleotides run in opposite directions on the two strands of each DNA molecule.

The phosphate group of each nucleotide is attached to the 5’ carbon of the deoxyribose of that nucleotide.
The phosphate group is also attached to the 3’ carbon of the next nucleotide in the string.
When the two strands are lined up to form the double helix, and base pair correctly, the orientation of one strand is upside-down compared to the other.
(See figure 16.12 in the text for a very helpful picture of this.)
One end of each strand is terminated by a 3’ carbon of its last, or terminal, deoxyribose with an –OH group hanging on the end.
The other end of each strand is terminated by a 5’ carbon of its terminal deoxyribose with a phosphate group attached.
Therefore, we speak of the DNA strands as oriented from 5’ to 3’ (for one of the strands) and 3’ to 5’ (for the partner).

The fact that the strands are anti-parallel creates some interesting complications for DNA replication.