Mechanism of DNA replication in bacteria
- The genome of E. coli consists of a single circular DNA molecule of approximately 4.6 x 106 nucleotide pairs.
- Each replisome in E.coli moves at a rate of 1000 nucleotides per second and it takes about 40 minutes to complete one round of replication.
- E.coli chromosome replicates by the bidirectional θ mode from a single replication origin.
- Consist of factors involved in DNA replication. Replication in E.coli requires not just a single DNA polymerase but 20 or more different enzymes and proteins, each performing a specific task.
- Replication machineries include replication enzymes: DNA polymerase, DNA helicases, DNA clamps, DNA topoisomerases and replication proteins: single-stranded DNA binding proteins (SSB)
- Replication machineries are also called replisomes or DNA replicase systems
Replication bubble is an unwound and open region of a DNA helix where DNA replication occurs.
The replication fork is a structure that forms during DNA replication. It is created by helicases, which break the hydrogen bonds holding the two DNA strands together.
The resulting structure has two branching “prongs”, each one made up of a single strand of DNA.
These two strands serve as the template for the leading and lagging strands.
Each active replication fork requires five basic components:
- Helicase to unwind the DNA
- Single-strand-binding proteins to keep the nucleotide strands separate long enough to allow replication
- The topoisomerase II (gyrase) to remove strain ahead of the replication fork
- Primase to synthesize primers with a 3’-OH group at the beginning of each DNA fragment
- DNA polymerase to synthesize the leading and lagging nucleotide strands.
Bacterial replication is organized in membrane – bound Replication factories
- Replication machineries are known as factories because they do not move relatively to template DNA.
- The bacterial chromosome (replicon) is circular and attached to the equatorial perimeter (membrane).
- The replication of a circular bacterial chromosome is highly organized.
- Once bidirectional replication is initiated at the origin, the two replisomes do not travel away from each other along the DNA. Instead, the replisomes are linked together and tethered to one point on the bacterial inner membrane, and the DNA substrate is fed through this “replication factory”
- The tethering point is at the center of the elongated bacterial cell.
The “factory” model of DNA replication hypothesizes a specific nuclear structure in which the molecular machinery for replication fork are brought together.
Replicated DNA is threaded through the replication factory as a loop. The parental DNA template is fed to the factory from sides.
- Once replication is terminated, the cell divides, and the chromosomes sequestered in the two halves of the original cell are accurately partitioned into the daughter cells.
- When replication commences in the daughter cells, the origin of replication is sequestered in new replication factories formed at a point on the membrane at the center of the cell, and the entire process is repeated.
The process by which newly replicated plasmids and chromosomes are actively segregated prior to cell division is called chromosome partitioning.
- After initiation, each of the two newly synthesized replication origins is partitioned into one half of the cell, and continuing replication extrudes each new chromosome into that half.
Replication in E.coli takes place in three stages: (Distinguished both by the reactions taking place and by the enzymes required).
Stage 1: Initiation of DNA replication
Stage 2: Elongation (Leading and lagging strand synthesis)
Stage 3: Termination of DNA replication
Primer removal, Gap filling and ligation
- DNA polymerase I removes the RNA primer using its 5’ to 3’ exonuclease activity.
- It then uses its 5’ to 3’ polymerase activity to replace the RNA nucleotides with DNA nucleotides (Gap filling).
How do they act?
DNA polymerase I attaches the first nucleotide to the OH group at the 3’ end of the preceding Okazaki fragment and then continues, in the 5’ to 3’ direction along the nucleotide strand, removing and replacing, one RNA nucleotide of the primer at a time.
- After polymerase I have replaced the last nucleotide of the RNA primer with a DNA nucleotide, a nick remains in the sugar – phosphate backbone of the new DNA strand.
(The 3’-OH group of the last nucleotide to have been added by DNA polymerase I is not attached to the 5’- phosphate group of the first nucleotide added by DNA polymerase III)
- This nick is sealed by the enzyme DNA ligase, which catalyzes the formation of a phosphodiester bond without adding another nucleotide to the strand
Regulation of DNA replication in E.coli
In E. coli, DNA replication is regulated through several mechanisms, including:
- The hemimethylation and sequestering of the origin sequence
- The ratio of adenosine triphosphate (ATP) to adenosine diphosphate (ADP)
- The levels of protein DnaA
(All these control the binding of initiator proteins to the origin sequences).
- In E.coli, Dam methylase enzyme methylates 5’ GATC sequence at the OriC. DNA synthesis results in hemimethylated sequences. This hemimethylated DNA is recognized by the protein SeqA, which binds and sequesters the origin sequence.
- In addition, DnaA (required for initiation of replication) binds less well to hemimethylated DNA. As a result, newly replicated origins are prevented from immediately initiating another round of DNA replication.
- ATP builds up when the cell is in a rich medium, triggering DNA replication once the cell has reached a specific size. ATP competes with ADP to bind to DnaA, and the DnaA-ATP complex is able to initiate replication.
- A certain number of DnaA proteins are also required for DNA replication — each time the origin is copied, the number of binding sites for DnaA doubles, requiring the synthesis of more DnaA to enable another initiation of replication.