DNA Replication in prokaryotes

DNA replication

DNA replication is an extraordinarily important and complex process upon which all life depends. During DNA replication, the two strands of the double helix are separated; each then serves as a template for the synthesis of a complementary strand according to the base-pairing rules. Each of the two progeny DNA molecules consists of one new strand and one old strand, and DNA replication is said to be semi-conservative. 
DNA Replication

DNA Replication Machinery

DNA replication is essential to organisms, and a great deal of effort has been devoted to understanding its mechanism. The replication of E. coli DNA requires at least 30 proteins. The overall process of DNA replication is similar in all organisms.

Enzymes called DNA polymerases catalyze DNA synthesis. All known DNA polymerases catalyze the synthesis of DNA in the 5' to 3' direction, and the nucleotide to be added is a deoxynucleoside triphosphate (dNTP ). Deoxynucleotides are linked by phosphodiester bonds formed by a reaction between the hydroxyl group at the 3' end of the growing DNA strand and the phosphate closest to the 5' carbon (the a-phosphate) of the incoming deoxynucleotide.

DNA Polymerase Reaction
The energy needed to form the phosphodiester bond is provided by release of the terminal two phosphates as pyrophosphate (P Pi) from the nucleotide that is added. The P Pi is subsequently hydrolyzed to two separate phosphates (PJ Thus the deoxynucleoside triphosphates dATP, dTTP, dCTP, and dGTP serve as DNA polymerase substrates while deoxynucleoside monophosphates (dNMP s: dAMP, dTMP, dCMP, dGMP ) are incorporated into the growing chain.

For DNA polymerase to catalyze the synthesis of DNA, it needs three things. The first is a template, which is read in the 3 ' to 5' direction and is used to direct the synthesis of a complementary DNA strand. The second is a primer (e.g., an RNA strand or a DNA strand) to provide a free 3 '-hydroxyl group to which nucleotides can be added. The third is a set of dNTP s. E. coli has five different DNA polymerases (DNA polymerase I-V). DNA polymerase III plays the major role in replication.

DNA polymerase III holoenzyme is a multifunctional enzyme composed of 10 different proteins. Most evidence suggests that within the complex are found two core enzymes Each core enzyme binds one strand of DNA and is responsible for catalyzing DNA synthesis and proofreading the product to ensure fidelity of replication. Associated with each core enzyme is a subunit called the 13 clamp. The 13 clamp tethers a core enzyme to the DNA. At the center of the holoenzyme, and represented by an octopus-like structure is a complex of proteins called the clamp loader, which is responsible for loading the 13 clamp onto DNA.
DNA Polymerase 3 Holoenzyme
A dimer of another protein (tau) holds the holoenzyme together. Because there are two core enzymes, both strands of DNA are bound by a single DNA polymerase III holoenzyme. DNA polymerase III holoenzyme is only one component of a huge complex of proteins called the replisome.

Other proteins found in the replisome include helicases, single-stranded DNA binding proteins, and topoisomerases. Helicases are responsible for separating (unwinding) the DNA strands just ahead of the replication fork, using energy from ATP hydrolysis. Single-stranded DNA binding proteins (SSBs) keep the strands apart once they have been separated, and topoisomerases relieve the tension generated by the rapid unwinding of the double helix (the replication fork may rotate as rapidly as 75 to I 00 revolutions per second).

Once the template is prepared, the primer needed by DNA polymerase III can be synthesized. An enzyme called primase synthesizes a short RNA strand, usually around 10 nucleotides long and complementary to the DNA; this serves as the primer.

Steps in the Replication Fork

In E. coli, DNA replication is initiated at specific nucleotides called the oriC locus (for origin of
chromosomal replication). This site is AT rich. Recall that adenines pair with thymines using only two hydrogen bonds, so AT-rich segments of DNA become single stranded more readily than do GC-rich regions. This is important for initiation of replication.
E.coli DNA replication

  1. The bacterial initiator protein DnaA is responsible for triggering DNA replication. DnaA proteins bind regions in oriC throughout the cell cycle, but to initiate replication, DnaA proteins must bind a few particular oriC sequences. The presence of DnaA at these sites recruits a helicase (usually DnaB helicase) to the origin.
  2. The helicase unwinds the helix with the aid of topoisomerases such as DNA gyrase. The single strands are kept separate by SSBs.
  3. Primase synthesizes RNA primers as needed. A single DNA polymerase III holoenzyme catalyzes both leading strand and lagging strand synthesis from the RNA primers.
  4. After most of the lagging strand has been synthesized by the formation of Okazaki fragments, DNA polymerase I removes the RNA primers. DNA polymerase I does this because, unlike other DNA polymerases, it has the ability to snip off nucleotides one at a time starting at the 5' end while moving toward the 3' end of the RNA primer.
  5. Finally, the Okazaki fragments are joined by the enzyme DNA ligase, which forms a phosphodiester bond between the 3 '-OH of the growing strand and the 5 '-phosphate of an Okazaki fragment.
DNA Replication Steps

Termination of Replication

In E. coli, DNA replication stops when the replisome reaches a termination site (ter) on the DNA. A protein called Tus binds to the ter sites and halts progression of the forks. In many other bacteria, replication stops spontaneously when the forks meet. Regardless of how fork movement is  stopped, there are two problems that often must be solved by the replisome. One is the formation of interlocked chromosomes called catenanes. 

The other is a dimerized chromosome-two chromosomes joined together to form a single chromosome twice as long. Catenanes are produced when topoisomerases break and rejoin DNA strands to easesupercoiling ahead of the replication fork.

The two daughter DNA molecules are separated by the action of other topoisomerases that break both strands of one molecule, pass the other DNA molecule synthesis occurs at each replication fork. When replication is complete, a circular molecule has been formed that is twice the length of the parent chromosome. Thus it is a dimerized chromosome. An enzyme called telomere resolvase (ResT) cuts the two chromosomes apart as it forms hairpin ends for each daughter molecule.


No comments