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10.2.4. The sense of growth of the new strands

The sense of growth of DNA

In vitro DNA synthesis

Kornberg, a disciple of the biochemist Severo Ochoa , studied in 1956 how a new complementary strand of DNA was synthesized from another strand, and which enzyme regulated replication. He isolated the DNA polymerase enzyme from Escherichia coli bacteria, and found that it is capable of synthesizing DNA in vitro .

The DNA polymerase requires deoxyribonucleotides -5-triphosphates adeninethymineguanine and cytosinemagnesium ions Mg2+, and that one of the chains of the DNA molecule made of "pattern" and an end acting as "primer”.

The DNA polymerase is an enzyme consisting of about 1000 amino acids, and is located in the nucleus and in the mitochondria. This enzyme binds the standard DNA, the primer DNA, and the nucleotide to be added. It can synthesize DNA from natural DNA in vitro (not in a living cell).

The DNA polymerase can not start the synthesis of a chain, but can only add nucleotides to the end of a chain preexisting, the DNA primer. It only adds the nucleotides at the end of the chain that the 3 'carbon of the nucleotide has free.

Thus, the strand that begins with the primer DNA only grows in the 5 '→ 3' direction. Thus, the nucleotide that has its free 5 'carbon is the first nucleotide of the DNA chain and the nucleotide that has free its 3' carbon is the last nucleotide that has been added to the chain, and to which another can be added.

The new synthesized chain is antiparallel and complementary .

The problem of direction in in vivo DNA duplication

Cairns, in 1963, carried out one of the first experiments that tried to observe how DNA replication developed.

He introduced bacteria Escherichia coli in a medium with thymine labeled with tritium (H3), that is, a thymine that had tritium instead of hydrogen. Tritium is a radioactive isotope that emits beta (β) particles, capable of impressing a photographic plate, which makes it possible to locate the new DNA molecules that have been synthesized.

Every few minutes the amount of thymine (with H3) contained in the bacterial DNA was checked, obtaining a sequence of images with the complete duplication of the DNA.

  • The first images were V-shaped , the next half-moons, and the last ones looked like more or less squashed circles .
  • The V-forms are the replication forks, formed by the two new DNA chains (with H3) that are synthesized on the DNA chains that have been opened to make templates.
  • The crescent shapes correspond to the replication bubbles.
  • The circular shapes made it possible to discover that the DNA of the Escherichia coli bacterium was circular.
  • With this experiment, in addition to confirming the semi - conservative hypothesis of DNA replication, it was also discovered that DNA replication occurred from a specific point. Although at that time, it was erroneously deduced that the replication was unidirectional, later it has been found that it is bidirectional , having a fork to the left of the starting point and another fork to the right, progressing in opposite directions.

By César Benito Jiménez [CC BY-SA 2.5 es or CC BY-SA 2.5 es], via Wikimedia Commons

But this fact raised two dilemmas:

How could I synthesize DNA polymerase without the need for a primer?

How could the two new chains grow in parallel?

If one strand grew in the 5'→ 3' direction, the other would have to grow in the 3'→ 5' direction, which is impossible to explain, since no DNA polymerase adds nucleotides in that direction.

The answer to these dilemmas was given by Okazaki in 1968. He discovered that there were some fragments made up of about 50 nucleotides of RNA and about 1000 or 2000 nucleotides of DNA, called Okazaki fragments . They were synthesized by RNA polymerase , which does not need any primer to start, and then by DNA polymerase , which added nucleotides in a 5'→ 3' direction on the master strand. Then the piece of RNA is lost and the rest of the fragments fuse, giving the impression that the strand grows in a 3'→ 5' direction. It is like it can happen in the entrance queue of a show, that although people walk forward, the queue grows backwards.

Fundamental ideas about the growth direction of new DNA strands

  • The DNA polymerase is an enzyme that is responsible for adding deoxyribonucleotides-5-triphosphates adeninethymineguanine and cytosine, following one strand of the DNA molecule that makes "pattern".
  • The DNA polymerase adds nucleotides, but it is necessary that there is a chain preexisting to add. You need a primer DNA to start adding nucleotides to it.
  • The DNA polymerase nucleotide added in a 5'→ 3' .
  • If one strand adds nucleotides in a 5'→ 3' direction, the other should do so in a 3'→ 5' direction, but DNA polymerase cannot. 
  • The RNA polymerase does not need a primer to add nucleotides of RNA to which the DNA polymerase can add nucleotides other DNA in the 5 '→ 3'.
  • The Okazaki fragments are short DNA strands newly synthesized in the discontinuous chain, synthesized in the 5'→ 3' primers from RNA which are then removed.