6.4.2 The Machinery and the Enzymes

In living cells, such as E. coli, the process of replication requires a set of catalysts (enzymes). The main enzyme is referred to as DNA-dependent DNA polymerase, since it uses a DNA template to catalyse the polymerisation of deoxynucleotides.

These enzymes are highly efficient enzymes as they have to catalyse polymerisation of a large number of nucleotides in a very short time. E. coli that has only 4.6 ×106 bp (compare it with human whose diploid content is 6.6 × 109 bp), completes the process of replication within 38 minutes; that means the average rate of polymerisation has to be approximately 2000 bp per second.

Not only do these polymerases have to be fast, but they also have to catalyse the reaction with high degree of accuracy. Any mistake during replication would result into mutations. Furthermore, energetically replication is a very expensive process. Deoxyribonucleoside triphosphates serve dual purposes.

In addition to acting as substrates, they provide energy for polymerisation reaction (the two terminal phosphates in a deoxynucleoside triphosphates are high-energy phosphates, same as in case of ATP). In addition to DNA-dependent DNA polymerases, many additional enzymes are required to complete the process of replication with high degree of accuracy.

For long DNA molecules, since the two strands of DNA cannot be separated in its entire length (due to very high energy requirement), the replication occur within a small opening of the DNA helix, referred to as replication fork. The DNA-dependent DNA polymerases catalyse polymerisation only in one direction, that is 5'Æ3'. This creates some additional complications at the replicating fork.

Consequently, on one strand (the template with polarity 3'Æ5'), the replication is continuous, while on the other (the template with polarity 5'Æ3'), it is discontinuous. The discontinuously synthesised fragments are later joined by the enzyme DNA ligase (Figure 6.8). The DNA polymerases on their own cannot initiate the process of replication.

Also the replication does not initiate randomly at any place in DNA. There is a definite region in E. coli DNA where the replication originates. Such regions are termed as origin of replication. It is because of the requirement of the origin of replication that a piece of DNA if needed to be propagated during recombinant DNA procedures, requires a vector.

The vectors provide the origin of replication. Further, not every detail of replication is understood well. In eukaryotes, the replication of DNA takes place at S-phase of the cell-cycle. The replication of DNA and cell division cycle should be highly coordinated. A failure in cell division after DNA replication results into polyploidy(a chromosomal anomaly). You will learn the detailed nature of origin and the processes occurring at this site, in higher classes.

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