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The transmission of chromosomal DNA from one generation to another generation is very crucial for the cell to propagate. And this can only be attained when the DNA is correctly duplicated, and each daughter cell has an equal distribution of genome. The process by which DNA makes copies of itself is known as replication. The replication process is continuous in every replicon from the start site, i.e., origin to the terminus. It is assisted by the movement of the replicating point known as the replication fork. 

DNA replication

There can be two ways of this replication: one is unidirectional, and the second is bidirectional replication.DNA replication happens in the synthesis phase of the cell cycle. When Watson and Crick proposed the structure of DNA as a double-helical structure, they also offered that the two strands of DNA would get separate, and each strand then will act as the template strand for the other newly synthesised strand. Hence, after the process of replication, each DNA molecule will have one parental strand and one newly synthesised strand. And this was called semi-conservative DNA replication. 

Replication process

DNA replication is a complex multistep process that requires a number of enzymes, protein factors and ions.  

Origin of Replication –

Replication begins at a particular region of DNA which has a particular nucleotide sequence called autonomic replicating sequence or ARS. It is called origin of replication or Ori. Prokaryotes have a single origin of replication while eukaryotes have several of them, e.g., 3500 in Drosophila.

DNA of prokaryotes functions as a single replicating unit called replicon. DNA of a eukaryotic possesses a number of replicons or replicating units .Replication proceeds on both sides from the Ori. This is called bidirectional replication. Unidirectional replication is rare.

Activation of Deoxyribonucleotides –­

There are four types of deoxyribonucleotides – dAMP, dGMP, dTMP and dCMP. With the help of energy, phosphate and an enzyme, phosphorylase, the nucleotides are changed into triphosphate state.

Initiation- 

Origin of replication is recognised by the origin recognition complex. It attracts enzymes. Enzyme helicase unwinds the DNA helix and unzips the two strands of DNA. The separated strands become stabilised in this condition with the help of single strand binding proteins or SSBPs.

Unwinding creates tension which is released by enzymes topoisomerases I and II. Topoisomerase II of prokaryotes is also called gyrase. It functions both as helicase and topoisomerase.Unzipping creates a Y–shaped configuration called replication fork.

Priming –

Replication fork exposes two different ends of the two DNA strands, 3’ end and 5’ end. At the free 3’ end of one strand and fork end of the second strand (with free 5’ end) a small RNA is synthesised with the help of enzyme primase.

The synthesised RNA is called RNA primer. It is 4–12 nucleotides long. RNA primer functions as the 5′ end of the new strand (to be synthesised).

Base Pairing –

The two separated DNA strands in the area of replication fork now function as templates.Their nitrogen bases attract complementary phosphorylated nucleotides, i.e., dATP opposite T, dTTP opposite A, dCTP opposite G, dGTP opposite C. Enzyme pyrophosphate removes two phosphates from phosphorylated nucleotides and changes them into monophosphate state. It releases energy which is used in building hydrogen bonds.

New Strand Formation –

  • It requires enzyme polymerase III in prokaryotes and polymerase d in eukaryotes alongwith source of energy ATP/GTP/TTP and Mg+2
  • The enzyme helps in establishing phospho–diester linkages between successive nucleotides. This produces polynucleotide chain.Polymerase III or d adds nucleotides only at 3’ end of the chain so that DNA strand formation occurs in 5’ → 3’ direction.
  • Since 3’ ends of the new chains would lie in opposite directions of the separated DNA template strands, chain elongation occurs in opposite directions.
  • The parent DNA strand unzippers further to expose new regions for chain building. However, while DNA synthesis is continuous in one strand, it is formed in small stretches in the other strand because of the opposite arrangement of nucleotides.
  • Where a new DNA strand is built in small segments, a new RNA primer is formed everytime in the region of opening of the chain.
  • RNA primers are removed by polymerase I in prokaryotes and polymerase a in eukaryotes. The same enzyme also extends the DNA strand in the region exposed by dissolution of RNA primer.
  • Replication is discontinuous over the strand over which only small stretches of DNA are built due to opposite running of DNA template. The small stretches of DNA are called Okazaki fragments named after the scientist Okazaki who discovered them in 1968.Okazaki fragments are joined together by enzyme DNA ligase (Khorana 1967).
  • New DNA strand built of Okazaki fragments is called lagging strand because it takes longer time to develop.The other replicated strand grows continuously and is called leading strand.
  • Since replication is continuous over one strand and discontinuous over the other, it is called semicontinuous.

Proofreading 

Though DNA polymerase is very accurate, a wrong nucleotide gets inserted occasionally. The incidence is 1 in 10,000. DNA polymerase–III is even unable to differentiate uracil from thymine.Mismatched nucleotides are removed and replaced by correct nucleotides with the help of enzyme DNA polymerase I and III.

Conclusion

Replication is the process in which a DNA molecule duplicates or makes a copy of itself. The DNA is duplicated when a cell divides. There is a formation replication fork as DNA unwinds. And from here, the replication process starts. The new helix will have the original DNA strand and the newly-formed strand of DNA. Hence the process of replication is very important for the growth of cells, repair and reproduction.