Enzymes and Mechanism of Replication

Carrying out replication accurately and quickly enough to keep pace with cell division requires a coordinated team of specialised enzymes. In prokaryotes, three distinct DNA polymerase enzymes are involved: DNA polymerase III is the principal enzyme responsible for the actual bulk synthesis of new DNA during replication, while DNA polymerase I, also called the Kornberg enzyme, and DNA polymerase II are chiefly involved in DNA repair rather than routine replication. Eukaryotic cells use five distinct types of DNA polymerase, all of which build a new strand by adding nucleotides exclusively onto the free 3'-OH end of the growing chain. Replication is extraordinarily fast given how little error it can tolerate -- E. coli, with about 4.6 million base pairs of DNA, completes an entire round of replication in only about 38 minutes, and any error introduced during this rapid copying would become a permanent mutation were it not for dedicated repair enzymes, such as nucleases, that continually proofread and correct mistakes as replication proceeds. The energy and raw material for building the new strand come from the same source: deoxynucleotide triphosphates (dNTPs) serve simultaneously as the substrate incorporated into the growing chain and as the source of the energy that drives the polymerisation reaction forward. Replication always begins at a specific initiation site on the DNA called the origin of replication (ori); prokaryotes, with their comparatively small, single circular chromosome, have just one such origin, whereas eukaryotes, whose much larger DNA molecules would take impractically long to copy from a single starting point, use many origins of replication (called replicons) spread across each chromosome, allowing replication to proceed from many points simultaneously. Because separating the two DNA strands along their entire length all at once would demand an enormous amount of energy, replication instead proceeds through a small, localised opening in the helix called the replication fork, with two such forks moving outward in opposite directions from each origin. Unwinding of the double helix at the fork is carried out by the enzyme DNA helicase, working together with topoisomerases (DNA gyrase in prokaryotes) that relieve the twisting strain this unwinding creates ahead of the fork. Because the two parental strands run in opposite (antiparallel) chemical directions, and DNA polymerase can only add new nucleotides onto a free 3'-OH end, extending a strand only in the 5' to 3' direction, the two new strands at a fork cannot both be synthesised the same way: on the strand whose template reads 3' to 5' in the direction the fork is opening, the new strand, called the leading strand, is built continuously in one smooth motion; on the other template strand, whose polarity runs the opposite way, the new strand, called the lagging strand, must instead be built discontinuously, in short stretches called Okazaki fragments, each one still s …