Describe the stepwise process of DNA replication in Escherichia coli

Replication begins at a specific origin of replication called oriC. The DnaA protein binds to DnaA boxes within oriC, causing the DNA to unwind locally. This unwinding is facilitated by ATP hydrolysis and creates an open complex. DnaC then loads the DnaB helicase onto the unwound DNA, forming the pre-priming complex.

DnaB helicase, powered by ATP, unwinds the DNA double helix in both directions from oriC, creating two replication forks. Single-strand binding proteins (SSBs) bind to the separated single strands, preventing them from re-annealing and protecting them from degradation. DNA gyrase (a type of topoisomerase II) relieves the positive supercoiling that builds up ahead of the replication forks due to unwinding.

Since DNA polymerase cannot initiate DNA synthesis de novo, an RNA primer is required. Primase (DnaG), an RNA polymerase, synthesizes short RNA primers (typically 10-12 nucleotides long) on both leading and lagging strands at the replication forks. These primers provide a free 3'-hydroxyl group for DNA polymerase to extend.

DNA Polymerase III (Pol III) is the primary enzyme responsible for synthesizing new DNA strands.

• Leading Strand Synthesis: On the leading strand, which is synthesized continuously in the 5' to 3' direction (following the replication fork), Pol III extends a single RNA primer.
• Lagging Strand Synthesis: On the lagging strand, which is synthesized discontinuously in the 5' to 3' direction (away from the replication fork), Pol III synthesizes short DNA fragments called Okazaki fragments. Each Okazaki fragment requires a new RNA primer. The replisome, a complex containing two Pol III enzymes, coordinates the synthesis of both strands.

Once an Okazaki fragment is complete, DNA Polymerase I (Pol I) removes the RNA primers using its 5' to 3' exonuclease activity. Simultaneously, Pol I fills the resulting gaps with deoxyribonucleotides using its 5' to 3' polymerase activity.

After Pol I has removed the primers and filled the gaps, a small nick remains between the newly synthesized DNA fragment and the adjacent Okazaki fragment (or the leading strand). DNA ligase then forms a phosphodiester bond, sealing these nicks and creating a continuous DNA strand.

Replication terminates when the two replication forks meet at specific termination (Ter) sites on the chromosome, located opposite oriC. Tus proteins bind to the Ter sites, acting as contra-helicases that block the progression of the DnaB helicase, thereby halting the replication forks. The two newly replicated circular chromosomes are often catenated (interlinked) and are then separated by topoisomerase IV.