DNA Replication: Prokaryotes.

DNA is the genetic material which has to be distributed to the daughter cells. Hence the entire DNA molecule should be replicated before the cell division.

DNA replication is an important process which takes place in every organisms, be it prokaryotic or eukaryotic. The DNA replication process produces two identical copies of daughter DNA molecules using the existing DNA molecule as template. Each daughter DNA molecule inherits one strand from the parent cell and the other strand is newly synthesized. This is known as semiconservative mode of replication, demonstrated by Meselson and Stahl (see the original paper here).

Replication process in eukaryotic and prokaryotic organisms share a lot similarity with few differences. In this post we discuss about the DNA replication in the prokaryotes.

The Stages in Replication:

The DNA replication in prokaryotes (and eukaryotes) are divided into three stages: initiation, elongation, and termination.

Initiation:

During initiation the different proteins bind and form complex called orisome at origin which unwinds the DNA. The different components and their functions are as follows;

• The origin:

The replication of DNA initiates at a particular sequence of nucleotides in the genome, known as origin. The prokaryotic cell usually has one origin as the genome is small and circular. The initial loading of the replication machinery known as orisome, a nuclear-protein complex, and the initial unwinding takes place at origin.

In E. coli chromosomal DNA replication initiates at origin of chromosomal DNA, oriC. The oriC is a 245-bp region containing four 9-bp and are three repeats of a 13-bp A/T-rich sequence.

(Just for info: Read this paper if want to know more about orisomes.)

• DnaA protein: It is also known as the initiator protein. This protein with 4 different domains is the first one to recognize and bind to the origin of replication at the 9 bp regions. It is the binding of the initiator protein that actually causes the DNA to stretch and leads to the separation of the strands at the AT-rich region. As the two strands separate more DnaA proteins bind the unwound DNA.

• Dna C helicase loader: These proteins interact with the ssDNA-bound DnaA proteins. Dna C helicase loaders help load the Dna B helicase onto the unwound DNA.

• Dna B helicase: Helicase continues the unwinding of the DNA.

• Single-stranded DNA binding protein (SSB): These proteins bind and stabilize the unwound DNA. Hence the unwound DNA do not form base pairs again.

• DNA gyrase: DNA gyrase is a topoisomerase that removes the twist resulting from the unwinding of the DNA, by cleaving a strand of the DNA helix, untwisting it and then resealing the broken strand again.

As the DNA is unwound, the area appears like a bubble (fig 1) and is known as a replication bubble.

The replication bubble has two Y shaped ends known as replication forks, (fig 2 shows one of the two replication fork) which is the junction between the wound ds DNA and ss unwound DNA.

The two unwound strands are complementary and each can serve as the templates for the synthesis of the new strands of DNA.

Elongation:

During elongation, the DNA pol III extends the daughter strands as the replication complex known as replisome travels along the length of the chromosome. The replisome includes a helicase, double-stranded DNA, a DNA polymerase(s) and a clamp loader.

• DNA Polymerase

The first DNA polymerase enzyme to be characterized, DNA polymerase I (pol I) from the bacterium Escherichia coli. DNA pol I is not the main DNA replicating enzyme but is involved in processing of RNA primers during lagging-strand synthesis and DNA repair mechanisms.

The major polymerase involved in the replication is DNA polymerase III, which is a dimer, with two similar multisubunit complexes.

In DNA pol III monomer is made up of core enzyme, γ complex and the β clamp.

is The DNA pol III core is made up of the αεθ complex, wherein α is the Polymerase subunit, ε is the exonuclease (proofreading) subunit, and θ is involved with the stabilizing.

The γ complex (γδδ′χψ) is required for the loading and unloading of the β clamps, the ring-shaped protein complexes that holds and slides along duplex DNA. β clamps allow the polymerase to remain associated with DNA and still slide along the DNA.

Each pol III monomer of the complex catalyzes the replication of either of the two DNA strands at the replication fork at the rate of around 1000 bp per second.

DNA polymerase enzymes join the phosphate group at the 5′ carbon of a new nucleotide to the hydroxyl (OH) group of the 3′ carbon of a nucleotide already in the chain. Because of this there are two needs that arise;

~ First the DNA pol needs an existing 3’OH end.

This problem is solved by creating RNA primer

• Dna G primase: As the replication bubble is formed, the Dna G primase initiates the synthesis of RNA primer, a sequence of about 10 RNA nucleotides complementary to the parent DNA template. RNA primer provides a free 3-OH end for the addition of the base pairs by the DNA polymerase. The RNA nucleotides in the primers are then replaced by DNA nucleotides.

~ Second the replication can only proceed in 5′→3′ direction.

Now as DNA polymerase III can add nucleotides only to the 3′ end of a DNA strand having OH group, the replication always proceeds only in the 5′ → 3′ direction on a growing DNA strand.

However, as the two parent strands of a DNA molecule are antiparallel, one strand is unwound from the 5′ end and other from the 3′ end at the replication fork (fig 4a).

In the strand which is unwound from the 5′ end (Fig 4b), the primer is synthesized, such that it’s 3′ end faces towards the unwinding replication fork. Hence the DNA pol III can continuously add nucleotides to the 3′ end (Fig 4c) and extend the DNA daughter strand (fig 4d). This strand which is synthesized continuously is known as the leading strand, in which the direction of daughter strand elongation is same as the direction of unwinding.

The other strand, known as lagging strand, experiences the unwinding from the 3′ end. The primase synthesizes the primer with 5′ end towards the unwinding end and the and the 3′ end is on the opposite side. Now the DNA pol III extends the daughter DNA strand at the 3′ end. Hence the unwinding of the parent duplex and extension of daughter DNA strand takes place in the opposite direction.

In this lagging strand the replication cannot be continuous as in the leading strand. The primase synthesizes the first primer (say P1 in fig 5b) and moves towards the 3′ end along the direction of replication fork. The DNA pol III adds nucleotides to the 3′ end of the primer as the replication fork moves in opposite direction. As the DNA is unwound, the region to the 5′ end of the primer (P1) remains unreplicated (please see fig 5c).

To replicate this region primase synthesizes another primer (P2) at the point closer to the replication fork (see fig 5c). Hence the new RNA primer is constructed and the DNA pol III jumps ahead to it and begin synthesis of the next DNA fragment (from the 3′ end of the primer P2 to the 5’end of the primer P1, as seen in fig 5d).

Hence the replication of the lagging strand takes place in discontinuously as a series of short fragments of DNA. These fragments are called Okazaki fragments, and are about 1000 to 2000 nucleotides long in prokaryotes.

As the Okazaki fragments are completely synthesized, the enzyme DNA polymerase I removes the RNA primer and fills in the gap. DNA pol I also fills in any gaps between Okazaki fragments following which the enzyme DNA ligase joins the adjacent Okazaki fragments to the lagging strand.

“Hence one of the important feature of Replication is that the synthesis of the leading strand is continuous, while that of the lagging strand is discontinuous.”

Termination:

Replication of the circular chromosome initiates at a origin, with two replication forks proceeding in opposite directions. The two forks move in the opposite direction around the circular chromosome and finally move towards the site opposite to the initiation site. This region is known as the replication terminus.

The replication terminus contains series of sites called Termination or ‘Ter’ sites. Ter sites sequences are recognized and bound by the Tus protein monomers.

The Tus–Ter complex is asymmetric and has one face that blocks replisome progression, the ‘nonpermissive’ face, and a second face that allows replication to continue, the ‘permissive’ face (Figure 1A) The non permissive face of Tus protein blocks DnaB helicase passage while permissive face permits the passage of the replisome complex.

When a replisome proceeds towards the nonpermissive face, a cytosine at position 6 of Ter site binds to the Tus protein. This strengthened association between Tus and Ter site, causes the replisome to disassemble from the DNA and single-stranded binding protein (SSB) binds and stabilises the ssDNA.

Later as the second replisome approaches the permissive face of the Tus–Ter complex, affinity of Tus for Ter site decreases and the Tus protein dissociates.

As the Tus protein dislodges, the replisomes replicates the the two strands, creating two complete daughter copies.

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References:

Allen et al. (2011) Roles of DNA polymerase I in leading and lagging-strand replication defined by a high-resolution mutation footprint of ColE1 plasmid replication. Nucleic Acids Res. 39(16): 7020–7033.

Pollard et al (2017) Chapt 42 – S Phase and DNA Replication. Cell Biology (Third Edition). 727-741.

Rudolph et al (2019) Termination of DNA Replication in Prokaryotes. 10.1002/9780470015902.a0001056.pub3.

Kaplan (2006) Replication Termination: Mechanism of Polar Arrest Revealed. Current Biology. 16(17): R684-R686.

Fijalkowska et al (2012) DNA replication fidelity in Escherichia coli: A multi-DNA polymerase affair. FEMS microbiology reviews. 36. 1105-21. 10.1111/j.1574-6976.2012.00338.x.

Beattie & Reyes-Lamothe (2015) A Replisome’s journey through the bacterial chromosome. Frontiers in Microbiology. 6. 10.3389/fmicb.2015.00562.