The Enzymes and Machinery of Replication

DNA replication is a highly coordinated process carried out by a large team of specific enzymes and proteins working together at a structure called the replication fork — the Y-shaped point where the parental double helix is actively being unwound and copied. The process begins at a specific site on the DNA molecule called the origin of replication, a sequence recognised by initiator proteins that assemble the rest of the replication machinery there. From this origin, the double helix is unwound in both directions, so replication typically proceeds bidirectionally, with two replication forks moving away from the origin in opposite directions simultaneously.

Unwinding the tightly wound double helix, and keeping the two separated strands apart long enough for new strands to be synthesised against them, requires several dedicated proteins. The enzyme DNA helicase moves along the DNA at the replication fork, breaking the hydrogen bonds holding the two parental strands together and physically unwinding the double helix ahead of the advancing fork. Because unwinding one region of a closed, coiled DNA molecule creates mechanical strain (over-winding, or positive supercoiling) further along the molecule, the enzyme DNA topoisomerase (also called DNA gyrase in bacteria) works ahead of the helicase, cutting and re-joining the DNA strands to relieve this torsional strain. Once the two parental strands are separated, single-strand binding proteins (SSBs) coat each of the exposed single strands, preventing them from prematurely re-annealing (re-pairing) with each other or folding back on themselves before new strands can be synthesised against them.

The central enzyme of replication itself is DNA polymerase, which synthesises a new DNA strand by adding nucleotides one at a time to a growing strand, always reading the parental template strand in its 3' to 5' direction and therefore always synthesising the new strand in the 5' to 3' direction. DNA polymerase has one crucial limitation, however: it cannot begin synthesising a brand-new strand entirely from scratch, but can only ADD nucleotides onto the free 3'-OH end of an already-existing strand — so every new strand of DNA requires a short starter sequence, called a primer, to be laid down first. This primer is a short stretch of RNA, synthesised by the enzyme primase, which provides the essential free 3'-OH end that DNA polymerase can then extend. …