Initiation

The initiation of DNA replication in E. coli and other prokaryotes begins at a specific location on the DNA called the origin of replication (oriC). Prokaryotes typically have a single origin due to their circular DNA structure. Within oriC, two types of specific DNA sequences, known as 9-mers and 13-mers, play crucial roles in the initiation process. These sequences are recognized by replication initiation proteins, enabling the precise and regulated beginning of DNA replication.

Key Sequences in oriC

1. 9-mers  are sequences that are 9 base pairs long and occur multiple times within the oriC region, where they serve as binding sites for DnaA proteins, which are initiator proteins essential for starting DNA replication. DnaA Binding: When DnaA binds to these 9-mer sequences, it triggers a conformational change that allows additional DnaA proteins to bind. This creates a complex that induces a localized unwinding of the DNA at nearby 13-mer sequences.

2. 13-mers are 13 base pairs long and are located adjacent to the 9-mer sites within oriC, wherein these sequences are AT-rich regions, meaning they contain a high proportion of adenine (A) and thymine (T) bases. AT-rich sequences are easier to unwind due to their weaker hydrogen bonding (only two hydrogen bonds between A and T, compared to three between G and C). 

   • DNA Unwinding: When DnaA binds to the 9-mer regions, it causes strain that opens or "melts" the AT-rich 13-mer regions, creating single-stranded DNA (ssDNA) that allows other replication proteins to enter, such as helicase (DnaB) and primase, to initiate the formation of the replication fork.

Steps of Initiation and Preparation for DNA Synthesis

1. Recognition of oriC: DnaA proteins bind to the oriC region, specifically to the 9-mers, causing a slight unwinding of the DNA at this site. This process requires ATP.

2. Helicase Loading: Once the origin is partially unwound, another protein, DnaC, assists in loading DnaB helicase onto each DNA strand. DnaB helicase further unwinds the DNA in both directions, creating two replication forks where DNA synthesis will take place.

3. Single-Strand Binding Proteins (SSB): As the DNA unwinds, SSB proteins bind to the single-stranded DNA (ssDNA) to stabilize it, preventing it from reannealing or forming secondary structures.

Unwinding and Primer Synthesis at the Replication Fork

Once the replication fork is established, enzymes prepare the template strands for the synthesis of new DNA.

1. Helicase Activity: DnaB helicase continues to unwind the double helix at the replication fork, separating the strands to create templates for the leading and lagging strands.

2. Topoisomerase Activity: As helicase unwinds the DNA, it introduces supercoiling tension ahead of the replication fork. Topoisomerase (specifically DNA gyrase in prokaryotes) alleviates this tension by creating temporary nicks in the DNA to release supercoiling, allowing the replication machinery to proceed smoothly.

3. Primase Activity: The enzyme primase (DnaG in E. coli) synthesizes short RNA primers on each template strand. These RNA primers provide a 3' hydroxyl (-OH) group, which is necessary for DNA polymerase to begin adding nucleotides and synthesizing the new DNA strands.

Through these coordinated steps, the oriC region enables the correct initiation and progression of DNA replication in prokaryotes, ensuring accurate and efficient duplication of the circular DNA molecule.

Elongation

Elongation is the phase where new DNA strands are synthesized as complementary copies of the original template strands.

1. Leading Strand Synthesis

• Continuous Synthesis: On the leading strand, which runs in the 3' to 5' direction relative to the replication fork, DNA polymerase III continuously synthesizes the new DNA strand in the 5' to 3' direction.

• No Need for Additional Primers: Since DNA polymerase III can add nucleotides in a continuous manner as the replication fork opens, only a single RNA primer is needed at the start of the leading strand.

2. Lagging Strand Synthesis

• Discontinuous Synthesis: The lagging strand runs in the 5' to 3' direction relative to the replication fork, meaning it must be synthesized in short fragments (known as Okazaki fragments) because DNA polymerase can only add nucleotides in the 5' to 3' direction.

• Priming Each Okazaki Fragment: As the replication fork progresses, primase synthesizes an RNA primer for each Okazaki fragment.

• Extension by DNA Polymerase III: DNA polymerase III then extends each RNA primer, synthesizing DNA in short fragments along the lagging strand.

• Multiple Primers Needed: Each Okazaki fragment requires a separate RNA primer, leading to a series of disconnected DNA segments on the lagging strand.

3. Proofreading

• 3' to 5' Exonuclease Activity: DNA polymerase III has proofreading ability, allowing it to detect and correct any incorrectly paired nucleotides.

• Error Correction: If an error is detected, DNA polymerase III removes the incorrect nucleotide and replaces it with the correct one, ensuring accuracy and high fidelity in DNA synthesis.

4. Primer Removal and Gap Filling

• Primer Removal: Once DNA synthesis on both strands is complete, the RNA primers from each Okazaki fragment on the lagging strand must be removed. DNA polymerase I removes these primers using its 5' to 3' exonuclease activity.

• Gap Filling: After primer removal, DNA polymerase I fills the gaps with DNA nucleotides, connecting the adjacent Okazaki fragments to create a more continuous DNA strand on the lagging side.

5. Ligation

• Sealing the Nicks: After DNA polymerase I fills the gaps, the lagging strand still has small nicks (breaks) between adjacent Okazaki fragments.

• DNA Ligase Action: DNA ligase joins these fragments by forming a phosphodiester bond between the 3' hydroxyl end of one fragment and the 5' phosphate end of the next, creating a continuous DNA strand.

Summary

1. Leading Strand: Synthesized continuously in the 5' to 3' direction by DNA polymerase III.

2. Lagging Strand: Synthesized discontinuously as Okazaki fragments, each requiring an RNA primer.

3. Proofreading: DNA polymerase III corrects errors to ensure accuracy.

4. Primer Removal and Gap Filling: DNA polymerase I removes RNA primers and fills gaps with DNA.

5. Ligation: DNA ligase seals the nicks between fragments to create a continuous DNA strand.

This process ensures that both leading and lagging strands are accurately and efficiently replicated.

Termination

Termination of DNA Replication in E. coli - Stepwise

1. Replication Forks Meet:

   • As replication progresses, the two replication forks, which started at the origin of replication (oriC) and moved in opposite directions, eventually meet on the opposite side of the circular chromosome.

2. Encounter with Termination (ter) Sites:

   • Ter Sites: These are specific DNA sequences located on the chromosome, which serve as signals for the replication forks to stop. The arrangement of ter sites on the chromosome guides the replication forks to terminate in a controlled manner.

3. Binding of Tus Proteins:

   • Tus (Termination Utilization Substance): Special proteins called Tus bind to the ter sites. Once bound, Tus proteins act like "roadblocks," preventing the replication forks from moving past the termination region.

   • One-Way Block: Tus proteins allow the replication machinery to move towards ter sites from one direction but block further movement when approached from the opposite direction, bringing replication to a halt at the ter sites.

4. Decatenation:

   • Linked DNA Molecules: After replication completes, the two circular DNA molecules are often linked or tangled together, a state known as catenation.

   • Topoisomerase IV Action: To separate the two DNA molecules, an enzyme called Topoisomerase IV creates a temporary break in one of the DNA strands, allowing it to unwind from the other molecule, and then reseals the break.

5. Separation of DNA Molecules:

   • After decatenation, each daughter DNA molecule is fully separated, ensuring they can be allocated into separate cells.

6. Result:

   • With two distinct, identical DNA molecules, each daughter cell will receive a complete chromosome during cell division.

This stepwise process ensures precise termination of replication and guarantees that each daughter cell inherits an accurate copy of the circular chromosome.