Mode of DNA Replication 

DNA replication is a multi step complex process which requires over a dozen enzymes. It begins at a particular spot called origin of replication. Bacterial and viral DNA has a single origin of replication but in eukaryotic DNA there are a number of origin of replication because of the large size as well as association with proteins. Replication takes place as follows : 

Activation of deoxyribonucleotides : Deoxyribonucleotides or deoxyribonucleoside monophosphates occur freely inside the nucleoplasm. They are first phosphorylated and changed to active forms which have three phosphate residues instead of one. Enzymes phosphorylase is required alongwith energy. The phosphorylated nucleotides are cfeATP. (deoxyadenosine triphosphate), deGTP (deoxyguanosine triphosphate), deCTP (deoxycytidine triphosphate) and deTTP (deoxythymidine triphosphate). 

Exposure of DNA strands : Enzy mes topoisomerases are specialized to cause nicks (or breaks) and reseal the same in one strand of DNA. Helicases (unwindases) unwind the DNA helix and separate the two strands. In prokaryotes topoisomerases and helicases are replaced by DNA gyrases. The separated strands are stabilized by helix destabilizing (HD) or DNA binding proteins (DNP). They become open for replication. However, whole of DNA does not open in one stretch but the point of separation proceeds slowly from one end to the other. It gives the appearance of Y-shaped structure called replication fork. 

RNA primer. It is essential for initiation of new DNA chains. RNA primer is a small strand of RNA which is synthesized at the 5′ end of new DNA strand with the help of enzyme called primase. It is specific. RNA primer is formed on the free end of one strand and fork end of the other strand. After start of nucleotide chain, RNA primer is removed and the gap is filled. 

Base pairing: The two separated DNA strands in the replication fork function as templates. Deoxyribonucleoside triphosphates come to lie opposite the nitrogen bases of exposed DNA templates – deTPP opposite A, deCYP opposite G deATP opposite T and deGTP opposite C. With the help of enzyme pyrophosphatase the two extra phosphates present on the deoxyribonucleotides separate. Energy is released in the process. The energy is used in establishing hydrogen bonds between the free nucleotides and nitrogen bases of templates. 

Chain formation : It requires DNA polymerase III (Komberg, 1956). In the presence of Mg²⁺, ATP (GTP), TPP and DNA polymerase III, the adjacent nucleotides present
attached to nitrogen bases of each template DNA strand establish phosphodiester bonds and get linked to form replicated DNA strand. As replication proceeds new areas of parent DNA duplex unwind and separate so that replication proceeds rapidly from the place of origin towards the other end. RNA primer is removed that the gap filled with complementary nucleotides by means of DNA polymerase I. Because of sequential opening of DNA double chain and its replication to form two chains. DNA replication is also called Zipper duplication. However, DNA-polymerase can polymerise nucleotides only in 5′ direction because it adds them at the 3′ end. Since the two strands of DNA run in antiparallel directions, the two templates prov ide different ends for replication. Replication over the two templates thus proceeds in opposite directions. One strand is formed continuously because its 3′ end is open for elongation. It is called leading strand. 

Replication is not continuous on the other template because only a short segment of DNA strand can be built in 5′ → 3′ direction due to exposure of a small stretch of template at one time. Short segments of replicated DNA are called Okazaki fragments. An RNA primer is also required every time a new Okazaki fragment is to be built. After replacing RNA primer the deoxv ribonucleotides and their polymerization. Okazaki fragments are joined together by means of enzyme. DNA ligase. DNA strand built-up of Okazaki fragments is called lagging strand. 

Proof-reading and DNA repair: A wrong base is sometimes introduced during replication. The frequency is one in ten thousand. DNA polymerase is able to sense the same. It goes back, removes the wrong base, allows addition of proper base and then proceeds forward. However, even DNA polymerase III is unable to distinguish uracil from thymine so that it is often incorporated in place of thymine. Such a mismatching is corrected by means of a number of enzymes. DNA replication is semi-conservative. One half of the parent structure passes up to each replica while the second half is built a new.