Part 5

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Do curso por University of Geneva

Classical papers in molecular genetics

95 classificações

University of Geneva

Classical papers in molecular genetics

95 classificações

You have all heard about the DNA double helix and genes. Many of you know that mutations occur randomly, that the DNA sequence is read by successive groups of three bases (the codons), that many genes encode enzymes, and that gene expression can be regulated. These concepts were proposed on the basis of astute genetic experiments, as well as often on biochemical results. The original articles were these concepts appeared are however not frequently part of the normal curriculum of biologists, biochemists and medical students. This course proposes to read study and discuss a small selection of these classical papers, and to put these landmarks in their historical context. Most of the authors displayed interesting personal histories and many of their contributions go beyond not only the papers we will read but probably all their scientific papers. Our understanding of the scientific process, of the philosophy underlying the process of scientific discovery, and on the integration of new concepts is not only important for the history of science but also for the mental development of creative science.

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Session 4

When DNA was found to be the genetic material, it was not known how this molecule could carry information. The structure of DNA thus became of critical importance. The available X-ray images obtained by M. Wilkins and R. Franklin only yielded a rough picture, and even R. Franklin, who had the clearest diffraction data, could not decide whether the molecules contained two or three strands. Both Pauling and Watson and Crick used molecular models with known inter-nuclear distances (bond length) and bond angles to predict a structure. While the model of Pauling was hardly realistic, since it used the protonated form of the phosphate, the model proposed by Watson and Crick proposed that DNA consists of a pair of DNA strands. Furthermore, it indicated that any nucleotide sequence could be accommodated in the structure. The only central biological issue that was addressed in the first paper was replication, and the famous sentence was really nothing more than a priority claim. Much more biology was discussed in the second paper. It was assumed that base pairing is sufficient to account for the fidelity of replication. The importance of DNA polymerase in replication fidelity was first demonstrated by Speyer.

Conheça os instrutores

Dominique Belin
Professor
Department of Pathology and Immunology University of Geneva Medical School

[MUSIC]

As we've seen, Watson and Crick proposed that mutations,

changes in the DNA sequence, are due to changes in the structure of the bases.

Well, that was a very, very simplified notion.

And in fact, what became clear later on, or it took about 12 years,

Was that the DNA preliminaries the enzyme, is in fact responsible for the fidelity.

Not the hydrogen bounce of the base payers.

This notion is based on one experiment, among others but on one,

the first experiment that showed that Was an experiment done by Speyer who was

Cold Spring Harbor, the lab which would be directed by Watson.

And he decided to test mutants in DNA polymerase

to see weather they effect fidelity of replication,

mutation frequency.

So he took mutants in DNA primaries of a phage, a phage T4 and

he took temperature sensitive mutants and checked them for the capacity

to induce mutations at a temperature at which the enzyme is still functioning.

Of course not at the temperature at which the enzyme is dead.

And he found with one of these prelimerates that this one,

L56, is capable of inducing

the reversion of a mutant into a Y type.

With a frequency of about a thousand fold higher than with the parent situation.

Another mutant, L141 is uncapabale of changing this reversion frequency.

So, depend when you change the DNA polymers you change the fidelity.

That notion was a strong departure from the Watson and Crick model.

Now, this notion led to the concept of geometric selection

put forward by Goodman and Eccles a few years later.

The fact that the enzyme is helping the plane to land on the carrier.

And you have to imagine that both are moving.

The notion of geometric selection is easily seen in this picture.

These are Watson-Crick base pairs.

A distance and an angle described first by Watson and Crick.

These three structures are base pairs with angles and

distances that are slightly different.

For instance, here, you have an angle of 69 degree, and

here you have an angle of 50 degree, roughly 50 degree, 51 degree.

Here, you have a distance of 10.3 angstrom versus 10.8 or 11.1.

Slightly different distance.

And the error is basically, this is possible, this is possible,

this is possible.

It's not made frequently because the preliminaries correct the landing

of the plane on the carrier.

Not the hydrogen bombs.

Because if you had only the hydrogen bombs,

you would form all these things at a certain frequency.

If you make a mistake, one of these mistakes doesn't change the number of

hydrogen bonds, you still have two two two hydrogen bonds.

So the polymerase is doing the job.

And now, the final hit to the hydrogen bond model.

Or hydrogen bond only model, was done still later, 97, not that long ago,

by people who managed to make a nucleotide that cannot form hydrogen bonds.

They replaced the oxygen by fluoride.

Same kind of electric properties.

Same size.

But cannot form hydrogen bombs.

They also replaced this this nitrogen by a carbon.

And when you don't write anything on this diagram, those are carbons.

This can make a hydrogen bond, this cannot.

Altogether, this defluorotoluene

cannot make hydrogen bonds, but looks like t, one of the bases of DNA.

This can be incorporated in front of an a and once it's incorporated into DNA,

it can serve as a template to incorporate an a.

So this modified t, incapable of forming hydrogen bond,

can be incorporated into DNA and can serve as a template.

So hydrogen bonds are not absolutely essential.

But we've seen the picture that Watson and Crick said.

The chains are held together by hydrogen bonds.

When in fact, there are several forces that together account for

the stability of double stranded DNA.

H bonds or b count.

But they're not as critical as generally admitted because it's easy to see them.

So it's easy to accept them.

The bases are hydrophobic.

They don't like water.

So they like to stick to each other because when they stick to each other,

water molecules can play together and enjoy themselves.

And water is, the energy of water playing is a major driving force in biology.

So the stacking of the base pairs,

one above each other, is a major force in making a stable double stranded DNA.

Or for that matter, a stable double stranded RNA.

The third force which is much more difficult to understand or

to grasp Is called the dipole moment.

The fact that each base has a certain electric gradient, like a little magnet.

The G has a negative charge on one side,

two negative charges, and one negative cell on the other.

The C has one positive charge here and one negative charge here.

So those are little magnets that run in almost opposite direction.

And little magnets attract each other.

Not like this, but like this.

In the case of at, it's much more difficult to find the magnet,

because the magnets are like this, and so they don't really attract or

repel each other, so there's not much effect.

For the at base pair, the stacking is critical and the h bonds are important.

So, what you see is that we've started with a double helix,

which was very simple.

There was an intrigue double helix, it was so simple that nobody really bothered

anymore to explain the energy that are involved.

But by doing this simplification, you forget two things.

You forget the role of enzymes in controlling fidelity,

which is a major issue.

And you forget the role of other forces in keeping things together.

And it's only if you think about that, that you can understand why sometimes

reactions don't behave the way you predict them to behave.

Because we always make assumptions.

Simplifying assumptions that are easy and

sometimes very often they work, but sometimes they just don't work.

It's nice to know what is making the molecules stick together?

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