Describe the process of DNA replication.

Explanation

The process of replication in living cells requires a set of enzymes and protein factors. DNA polymerase is the main enzyme. It is highly efficient to polymerise about 2000 bp per second. The whole machinery of DNA replication with all the enzymes is collectively called replisome. DNA replication in eukaryotes is semiconservative, semi- discontinuous and bidirectional as compared to semi-conservative, bidirectional and continuous in prokaryotes. DNA replication involves the following steps
(i) Origin of replication DNA replication begins at certain fixed unique points called origin. At origin, replication begins with a nick or incision made by an incision enzyme, l.e. endonuclease.
(ii) Activation of deoxyribonucleotides li occurs freely inside the nucleoplasm.
Deoxyribonucleotides are first phosphorylated and changed to active forms which have three phosphate residues instead of one. These triphosphates acts as substrate and provides energy for the polymerisation of nucleotides.
(iii) Unwinding of DNA helix DNA double helix is unwinded by enzyme helicase. The separated strands are stabilised by Single Stranded Binding Proteins (SSBPs). The unwinding of DNA imposes tension on the distal end of DNA molecule. This tension is released by the enzyme topoisomerase. They cause nicking and resealing the DNA strand. Alongwith topoisomerase, bacteria possess another enzyme called DNA gyrase, which can introduce negative super coils. Unwinding of DNA helix into two strands results in the formation of Y-shaped structure, called replication fork.
(iv) Formation of primer strand Initiation of DNA synthesis always requires a smaller segment of RNA called RNA primer (small strand of RNA). It is synthesised at 5 end of new DNA strand with the help of DNA specific RNA polymerase enzyme called primase. Formation of RNA primer constitutes the initiation phase of DNA synthesis, DNA polymerase cannot add nucleotides in the absence of RNA primer. Once the initiation of DNA synthesis is completed, this primer RNA is removed enzymatically.
(v) Addition of deoxyribonucleotides DNA polymerase progressively adds deoxyribonucleotides to the `3.rarrOH` end of the newly synthesised polypeptide chain.
(vi) Activity of DNA polymerase Two strands of a double helix are antiparallel to each other, i.e. one runs in the `5.rarr3.` direction, while the other has opposite `3.rarr5.` polarity.
DNA polymerase-Ill synthesises DNA in `5.rarr3.` direction only. This means that the strand with `3.rarr5.` polarity serves as a template for continuous DNA synthesis and called leading strand.
Other strand with 5 3. polarity forms the loop structure. This loop inverts the orientation of the template. DNA strand that is synthesised on this loop is fragmented or discontinuous and known as lagging strand or Okazaki fragments.
(vii) Elongation of new strand The enzyme DNA polymerase-l degrades the RNA primer and simultaneously catalyses the synthesis of short DNA segments to replace the primer. This segment is then joined to main DNA strand by DNA ligase enzyme.
(viii) Proofreading and DNA repair Sometimes during replication a wrong base is introduced erroneously. To compensate such inaccuracies, DNA polymerase possesses `3.rarr5.` exonuclease activity. Thus, it detects and excises a mismatched nucleotide. This process is known as proofreading.