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:
- Binding of DnaA protein to OriC
- Loading of DNA helicase
- Helicase opens the helix and binds primase to form primosome
- Synthesis of RNA primer
- 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.
