You prepare bacterial cell extracts by lysing the cells and removing insoluble d

Question

You prepare bacterial cell extracts by lysing the cells and removing insoluble debris via centrifugation. These extracts provide all the protiens required for DNA repication. The DNA template is a small, double-stranded circular piece of DNA (a plasmid) with a single origin of replication and a single replication termination site. The termination site is on the opposite side of the plasmid from the origin. Replication proceeds in both directions from the origin.

In addition to the extracts and the and the plasmid DNA, are there any additional materials you should add to this in vitro replication system? Explain.

Explanation / Answer

In order for plasmids to replicate independently within a host cell, they must possess the following -

A stretch of DNA that can act as an origin of replication (ori site).

The self-replicating unit, in this case the plasmid, is called a replicon. A typical bacterial replicon may consist of a number of elements, such as the gene for plasmid-specific replication initiation protein (Rep), repeating units called iterons, DnaA boxes, and an adjacent AT-rich region also is required.

Smaller plasmids make use of the host replicative enzymes and make copies of themselves, while larger plasmids may carry genes specific for the replication of those plasmids. A few types of plasmids are also capable of inserting into the host chromosome, and these integrative plasmids are called episomes.

Plasmid replication can be divided into three important stages: tey are initiation, elongation, and termination. The inability of DNA polymerases to initiate de novo replication makes necessary the independent generation of a primer. This is solved, in circular plasmids, by two main strategies: (i) opening of the strands followed by RNA priming or (ii) cleavage of one of the DNA strands to generate a 3?-OH end (rolling-circle replication).

Initiation is catalyzed most frequently by one or a few plasmid-encoded initiation proteins that recognize plasmid-specific DNA sequences and determine the point from which replication starts (the origin of replication). In some cases, these proteins also participate directly in the generation of the primer. These initiators can also play the role of pilot proteins that guide the assembly of the host replisome at the plasmid origin.

Elongation of plasmid replication is carried out basically by DNA polymerase III holoenzyme (and, in some cases, by DNA polymerase I at an early stage), with the participation of other host proteins that form the replisome.

Termination of replication has specific requirements and implications for reinitiation, studies of which are under progress.

all the requirements mentioned above play an important role in plasmid replication invitro.

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