Initiation of Replication and Template Preparation
Duplication of the bacterial chromosome cannot start at a random site. The assembly site for the replisome is a specific sequence called the origin (oriC). Specific nucleotide sequences in this region facilitate the initial melting of the double helix. From this region, one or two replication forks move outward until the entire molecule is duplicated.
An array of proteins coordinates the separation of the parental DNA strands:
- Helicase — an enzyme that physically unwinds the double helix at the replication fork, utilizing ATP hydrolysis.
- Topoisomerase (DNA gyrase) — relieves structural strain and prevents excessive supercoiling ahead of the replication fork.
- SSB proteins (Single-Strand Binding proteins) — rapidly bind to exposed single-stranded DNA, preventing the strands from reannealing and protecting them from nucleases.
Biochemical Mechanism of Synthesis and the Role of the Primer
DNA polymerase catalyzes the direct synthesis of the new strands. However, this enzyme has a fundamental biochemical limitation: it can only add new nucleotides to an existing 3'-hydroxyl group (3' end) of a preexisting chain. DNA polymerase cannot initiate synthesis de novo.
Initiation requires a specialized RNA primer complementary to the DNA template. DNA primase (an RNA polymerase) synthesizes this short RNA primer. Primase is unique because it can initiate RNA synthesis on a single-stranded DNA template without requiring a pre-existing 3'-OH group. Consequently, ribonucleotides are always initially present at the 5' end of each nascent strand before DNA polymerase extends the chain.
Asymmetry of the Replication Fork
Because the two strands of the DNA molecule are antiparallel, and synthesis can only proceed in the 5' to 3' direction, the two template strands are replicated differently at the replication fork:
- Leading strand. The direction of synthesis matches the movement of the replication fork. DNA polymerase follows the helicase continuously, synthesizing a long, unbroken strand.
- Lagging strand. The direction of synthesis is opposite to the movement of the replication fork. Synthesis must proceed discontinuously in short segments known as Okazaki fragments.
Maturation of the Lagging Strand and Cell Division
To become a continuous molecule, the lagging strand must undergo maturation. RNA primers are removed from each Okazaki fragment. DNA polymerase fills the resulting gaps with deoxynucleotides, and DNA ligase seals the nicks to form a continuous covalent strand.
Completion of chromosomal replication triggers bacterial cell division. Unlike eukaryotes, bacterial nucleoid segregation occurs without a mitotic spindle. Newly synthesized DNA strands attach to the cytoplasmic membrane. A spatial relationship exists between these attachment sites and the zone of active cell wall growth. As the membrane elongates, it physically separates the two anchored genome copies toward the poles of the future daughter cells. This binary fission ultimately drives exponential population growth.