Unit – III A
Replication of DNA
- The process in which a DNA molecule produces exact copy or replica of itself. is known as replication of DNA.
- In eukaryotes, replication of DNA takes place inside the nucleus.
- In DNA replication, the double helix (parent strand) unzips forming two separate strands called templates. These templates provide the base sequences used to synthesize new DNA (daughter) strands.
- Replication is a very complicated enzyme-catalyzed process. Enzymes are needed to unwind the DNA prior to replication and repackage the DNA after synthesis.
- There are three hypothesis proposed for replication of DNA
- In this process, the original helix serves as a template. The original molecule of DNA is preserved and entirely a new double-stranded molecule is synthesised.
- In this hypothesis, it is proposed that the original molecule is broken into fragments. Each fragment serves as a template for the synthesis of complementary fragments and finally, two new molecules are formed which consist of both old and new fragments.
- This hypothesis was proposed by Watson and Crick on the basis of their model of DNA molecule. They proposed that the DNA molecule untwists and start separating at one end.
- During this process, the covalent hydrogen bonds between complementary bases are broken.
- The separated strands act as a template and using thesis template two new molecules of DNA are synthesised
Experimental Evidence of Semiconservative Replication of DNA:
- In 1957, Matthew s. Meselson and Franklin W. Stahl confirmed semiconservative replication of DNA experimentally.
- They grew two cultures of E. coli in a medium using ammonium ions (NH4+) as the source of nitrogen for DNA (as well as protein) synthesis.
- One culture was developed on a medium of (NH4+) containing a light isotope (common) of nitrogen 14N and another culture was developed on a medium of (NH4+) containing a heavy isotope (rare) 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 could happen with either semi-conservative or dispersive replication. Thus the possibility of conservative replication was eliminated.
- This shows 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: a) half the DNA was normal and b) half was intermediate.
- As this experiment was performed for next generations, the density of DNA molecule decreased continuously. i.e. lighter and lighter molecules of DNA are formed. It was continuous decrease and not a random change. This eliminates the possibility of dispersive replication.
- It is evident from the experiment 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.
- Each daughter DNA molecule is one-half “old” and one-half “new”. Hence this replication is called semiconservative replication. It shows that the DNA strand is immortal. it will continue to serve as an unchanging template down through the generations.
Diagrammatic Comparision Between Three Hypothesis: