Termination of DNA replication in prokaryote (E.coli)
- In prokaryotes such as E.coli chromosomal DNA exists in a circular fashion whereby DNA replication begins at OriC. Two replication forks move bidirectionally from oriC to replicate DNA until they eventually meet, and the forks fuse with one another to form two circular daughter chromosomes.
- The region where the two replication forks meet is defined as the “terminus region” located roughly opposite of oriC.
- Bacteria use a “replication fork trap” system for successful termination of replication and fork fusion. This requires two factors:
1) DNA terminator (Ter) sites
2) A specific terminator protein that can bind Ter (Tus protein in E.coli)
DNA terminator sites (Ter)
- cis-acting DNA sequences. The Ter sequences are 23 bp non – palindromic sequences and are binding sites for the Tus protein.
- These elements are situated in the terminus region, approximately opposite to the origin of replication.
- Termination region (ter) of E.coli incorporates 10 Ter sites designated as TerA, TerB, …, TerJ. (5 of them are oriented in one direction and other 5 are oriented in opposite direction)
- TerC, TerB, TerF, TerG and TerJ are oriented to block the clockwise movement of replication forks
- TerA, TerD, TerE, TerI and TerH block the anticlockwise movement of replication forks.
- Each Ter site can interact with Tus
- Ter sites stall replication forks only when bound by proteins Tus in E.coli.
Terminator protein Tus (stands for terminus utilization substance)
- Product of the tus gene which is required for termination.
- Trans – acting replication termination protein binds to a specific Ter
- Acts as a counter-helicase when it comes in contact with an advancing helicase.
How Tus – Ter complex terminates DNA replication?
- The arrest of DNA replication in Escherichia coli is triggered by the encounter of a replisome with a Tus protein-Ter DNA complex.
- This DNA-protein complex arrests the progression of replication forks.
- The Tus-Ter complex acts by blocking the action of the DnaB helicase
- The Ter bound Tus protein effectively halts DNA polymerase movement.
The Ter sequences are positioned on the chromosome in two clusters with opposite orientations. The binding of Tus to ter elements provides a trap for the proceeding replication fork.
Depending on their orientation, these Tus bound sequences trap the replication fork. (They have functional polarity)
Because of the orientation of Tus bound Ter sites, each replisome that reaches the Ter region must cross all the Tus – Ter sites that are oriented the opposite way before arriving at a site that causes termination.
Most of the replication forks are trapped in the region between TerC and TerA. If the clockwise replication fork is delayed during DNA replication, termination occurs at TerA site. If the anticlockwise replication fork is delayed during DNA replication, termination occurs at TerC site
- Tus –Ter complex is specific for trapping the fork passing in one direction only. So a replication fork can pass through a Tus-Ter complex when traveling in one direction but not the other. Depending on that Fork progression is divided into two types.
Permissive Fork progression
The Ter-Tus protein complex responsible for catching the clockwise replication fork will allow the anticlockwise fork to proceed unchecked, until it is stopped by its own anticlockwise facing Ter element fork trap
Explanation for the statement
When a replication fork moving in clock wise direction encounters Tus – Ter complex oriented to trap counter clock wise moving replication fork, the Tus protein will dissociate from the complex and fork will continue its movement until it reaches the clock wise fork trapping Ter – Tus complex (Similarly fork moving in anticlockwise direction will not stopped by clockwise fork trap complex)
TerA sequences are oriented in such a way to trap the fork moving in counter clock wise direction. TerC sequences are oriented in such a way to trap the fork moving in clock wise direction. If clock wise moving fork reaches the TerA – Tus complex, fork movement will continue and replication progresses. This site is permissive for clock wise moving replication fork.
Like that If counterclockwise moving replication fork reaches the TerC – Tus complex, it will bypass them and replication continues.
Non – permissive fork progression
When a replication fork moving in clock wise direction encounters Tus – Ter complex oriented to trap clock wise moving replication fork, the fork progression will be arrested and the replication halts. This complex will prevent the helicase activity of DnaB protein.
A replisome moving in the clockwise direction shown by the arrow must first cross terE, terD, and terA before terminating replication at either the terC or terB site.
This arrangement ensures that each replisome continues to synthesize DNA until it collides with a replisome entering the Ter region from the opposite direction leading to the dissociation of both replisomes from DNA.
Biological roles of replication fork traps
- Prevention of over-replication
- The optimization of post-replicative mechanisms of chromosome segregation, such as that involving FtsK in Escherichia coli.
Resolution of the replicated chromosomes occurs when the two replication forks meet. Since these are moving in opposite directions, the distribution of ter sites roughly opposite to the ori ensures that the two replication forks will meet in the zone between the oppositely oriented ter sites.
Figure 3: Resolution of replication forks in the termination zone. (The abbreviations are cw=clockwise and ccw=counter-clockwise, lead=leading strand, lag=lagging strand).
Because bacteria have circular chromosomes, termination of replication occurs when the two replication forks meet each other on the opposite end of the parental chromosome.
One scenario is illustrated in this figure.
- Fork 1 moving in a counter-clockwise direction proceeds as far as it can (i.e. to the terD, terA sites).
- Fork 2, moving in a clockwise direction, can proceed past these ter sites, and it will meet Fork 1.
- The two sets of products from each replication fork are then joined. The leading strand synthesized from Fork 2 joins the lagging strand synthesized from Fork 1. Likewise, the lagging strand from Fork 2 joins the leading strand from Fork 1.
Decatenation: Replication of a closed circular chromosome results in two topologically-linked daughter chromosomes. The process of separating this linked daughter chromosome after replication is referred to as Decatenation.
- Separation of the catenated circles in E. coli requires topoisomerase IV (a type II topoisomerase). The separated chromosomes then segregate into daughter cells at cell division.
- (Transiently break both DNA strands of one chromosome and allowing the other chromosome to pass through the break).