Explanation of DNA Replication Process in E. coli

Introduction:

• DNA replication is the process by which cells copy their genetic material before cell division. In E. coli, DNA replication begins at a specific origin of replication called oriC. The process of DNA replication can be divided into three main stages: initiation, elongation, and termination.

Initiation

Binding of DnaA protein to OriC:

• DNA replication in E. coli starts with the binding of the DnaA protein to the OriC, the origin of replication.
• DnaA protein recognizes and binds to the 9-mers sequences present in the OriC, forming an initial complex.

Formation of the open complex:

• The binding of DnaA protein to the OriC 9-mers sequences facilitates the initial strand separation, or “melting,” of E. coli duplex DNA, which occurs at the OriC 13-mers.
• This process requires ATP and forms an open complex.

Loading of DNA helicase:

• Further melting of the two strands of the E. coli chromosome to generate unpaired template strands is mediated by the DNA protein, a helicase.
• One molecule of DnaB, a hexamer of identical subunits, clamps around each of the two single strands in the open complex formed between DnaA and OriC.
• This binding requires ATP and the dnaC protein.

Synthesis of RNA primer:

• The primers used during DNA replication in both prokaryotes and eukaryotes are short RNA molecules whose synthesis is catalyzed by the RNA polymerase primase.
• After the bound premises synthesize short primer RNAs complementary to both strands of duplex DNA, they dissociate from the single-stranded template.

The sequence of events during initiation:

1. Binding of DnaA protein to OriC
2. Loading of DNA helicase
3. Helicase opens the helix and binds primase to form primosome
4. Synthesis of RNA primer
5. Initiation of DNA polymerization by DNA polymerase

Elongation

Synthesis of DNA chain:

• DNA polymerases catalyze the step-by-step addition of deoxyribonucleotide units to a DNA chain.
• The chain-elongation reaction catalyzed by DNA polymerases is a nucleophilic attack by the 3′-hydroxyl group of the primer on the innermost phosphorus atom of the deoxyribonucleoside triphosphate.
• A phosphodiester bridge forms with the concomitant release of pyrophosphate.

Catalytic metal ions:

• The two catalytic metal ions present in the active site play an important role.
• Metal ion A interacts with the 3’OH, reducing the association between the O and the H. This leaves a nucleophilic 3’O.
• Metal ion B interacts with the triphosphates of the Incoming dNTP to neutralize their negative charge.
• After catalysis, the pyrophosphate product is stabilized through similar interaction with metal ion B.

Synthesis of leading strand:

• Elongation of the DNA chain proceeds in the 5′-to-3′ direction.
• At each growing fork, one strand, called the leading strand, is synthesized continuously from a single primer on the leading-strand template and grows in the 5′-3′ direction.
• Growth of the leading strand proceeds in the same direction as the movement of the growing fork.

Synthesis of lagging strand:

• Synthesis of the lagging strand is more complicated because DNA polymerases can add nucleotides only to the 3′ end of a primer or growing DNA strand.
• Movement of the growing fork unveils the template strand for lagging-strand synthesis in the 5′-3′ direction.
• After 1000 to 2000 nucleotides.

Termination

Bidirectional Replication of Bacterial Genomes

• Bacterial genomes are replicated bidirectionally from a single point.
• Two replication forks meet at a position diametrically opposite the origin of replication on the genome map.

Terminus Region Containing Ter Sequences

• Replication of genome terminates at terminus region.
• Terminus region contains multiple copies of about 23 bp sequences called Ter sequences.

Tus Proteins as Ter Sequence-Specific DNA-Binding Proteins

• Tus proteins are sequence-specific DNA-binding proteins that recognize Ter sequences.
• Seven Ter sequences have been identified in the E. coli genome.

Tus Proteins Block Replication Fork Progression in One Direction

• When bound to a terminator sequence, a Tus protein allows a replication fork to pass if the fork is moving in one direction.
• Tus protein blocks the passage of the DnaB helicase when approached from one direction.

DnaB Helicase Passes Tus Proteins from Opposite Direction

• DnaB helicase is responsible for the progression of the replication fork.
• DnaB can cross the Tus protein when approaching from the other direction.

Orientation of Termination Sequences Traps Replication Forks

• Orientation of the termination sequences and bound Tus proteins in the E. coli genome is such that both replication forks become trapped within a relatively short region on the opposite side of the genome to the origin.

Termination Occurs at or near Same Position

• Trapping of replication forks ensures that termination always occurs at or near the same position.

Proofreading

DNA Replication Accuracy

• DNA replication is very accurate with only about one error for every billion bases incorporated.
• Accuracy is necessary to keep the mutation load at a tolerable level, especially in large genomes.

DNA Proofreading

• DNA proofreading involves scanning the termini of nascent DNA chains for errors and correcting them before continuing chain extension.

3’→5′ Exonuclease Activity of DNA Polymerases

• 3’→5′ exonuclease activity is built into DNA polymerases.

Clipping Off Unpaired or Incorrectly Paired Bases at 3′ End of Primer

• When a template-primer DNA has a terminal mismatch, the 3’→5′ exonuclease activity of the DNA polymerase clips off the unpaired base or bases.

5’→3′ Polymerase Activity of Enzyme for Resynthesis

• When an appropriately base-paired terminus is produced, the 5’→3′ polymerase activity of the enzyme begins.