This is a unique process of making an identical copy of a double-stranded DNA, using existing DNA strand as a template for the synthesis of new DNA strands is called DNA replication.
Meselson and Stahl (1958) proved that DNA replicates by the semi-conservative methods by experimenting on E.coli.
- They used the heavy isotopes of nitrogen, i.e., 15N. They grew bacterial cells having DNA labelled with 15Nin 14N medium and found that F1 generation has DNA density intermediate between the two.
- Thus, the newly synthesized DNA possesses one strand contributed by parent DNA and other newly synthesized.
- Replication of DNA is energetically highly expensive. It requires a set of enzymes, mainly DNA dependent DNA polymerase.
- These enzymes catalyze the polymerization of a large number of nucleotides very accurately in a very short time.
- Replication requires abundant energy that comes from the breakdown of triphosphates of deoxyribonucleotides.
Steps for DNA Replication
- Enzymes helicase acts over the “ori” site and unzips (unwinds) the two strands of DNA. The separated strands are stabilized using Single-Stranded Binding Proteins (ssBPs).
- Unwinding creates tension in the uncoiled part by forming more supercoils. Tension is released by enzymes topoisomerases. They cause nicking of one strand of DNA (for removing coils) and resealing the same.
- The whole of DNA does not open in one stretch due to very high energy requirement, but the point of separation proceeds slowly towards both the direction. It gives the appearance of the Y-shaped structure called the replication fork.
- A small strand of RNA, called RNA primer, is synthesized at the 5′ end of the new DNA strand. It is essential for the initiation of new DNA chains as it is required by DNA polymerase to add nucleotides.
- DNA polymerase catalyses polymerization only in one direction, that is 5′ – 3′. Consequently, on one strand (the template with polarity 3- 5), the replication is continuous, which is called the leading strand.
- While on the other (the template with polarity 5′ – 3′), replication is discontinuous, which is called a lagging strand.
- Discontinuous replication occurs on lagging strand because the DNA polymerase enzyme catalyzes replication only in a 5′ – 3′ direction. Due to this, fragments are formed on this strand, which is called Okazaki fragments.
- The discontinuously synthesized fragments (Okazaki fragments) are later joined by the enzyme DNA ligase.
- The result of DNA replication is two DNA molecules consisting of one new and one old chain of nucleotides.
- This is why DNA replication is described as semi-conservative, half of the chain is part of the original DNA molecule and half is the newly synthesized chain.