This paper looks at how features of prokaryotic replication are essential for the prokaryote to replicate. Some of the features that will be discussed are initiation, replication and termination. Many references will be made to Escherichia coli which are prokaryote cells and its replication process has been studies extensively.
Deoxyribonucleic acid (DNA) contains genetic information. DNA functions to maintain the genetic material over decades which enable it to be passed down to the next generation. The information is then used to construct proteins and RNA amongst other cell components. The structure of DNA is composed of nucleotides which are further divided into nitrogenous bases, phosphate group and a Deoxyribose sugar (fig.1). The backbone of DNA is composed of Deoxyribose and phosphate group which are linked via phosphodiester linkages. Depending on where this linkage is on the Deoxyribose sugar indicates what directionally the strand has. The leading strand is arranged 3' to 5', showing that the phosphodiester linkages are between the 5th carbon and 3rd carbon on the Deoxyribose sugar, the lagging strand (antiparallel to the leading strand) has linkages between the 5' and 3' carbon. Nitrogenous bases can be divided into pyrimidines (cytosine, thymine and uracil) and purines (adenine and guanine). The unique shape of the bases allows them to fit into one another forming three hydrogen between cytosine and guanine and two hydrogen bonds between thymine and adenine (fig.1). The strong covalent bonds between the sugar-phosphate backbone and weak hydrogen bonds between bases gives arise to a double helix structure (fig.2). The complete structure of DNA in a prokaryote is circular, in addition to also being super coiled.
DNA replication in e-coli initiates at a single point called the Oric, which consists of 245 bases. It contains repetitive 13 mers which are made up of a high amount of Adenine and thymine bases joined by only 2 hydrogen bonds in contrast to cytosine and guanine which are joined by three hydrogen bonds, providing a weak point. The Oric also contains a 9 mers region where DnaA binds to causing the 13 mers region to unwind, separating the two strands into bubbles and exposing two asymmetrical replication forks at each end. Helicase now breaks the 2 hydrogen bonds joining adenine and thymine, and three bonds between cytosine and guanine, in addition to also unwinding the DNA by hydrolysing ATP. Single stranded binding proteins binds to the template strand preventing the two strands from reannealing. Topoisomerase gives extra support the Helicase by undoing the supercoiling past the replication forks in order for Helicase to unwind the DNA. DNA polymerase III now adds nucleotides consisting of deoxyribose sugar, phosphate group and complementary bases to the leading strand in the 5' to 3' direction joining adenine and thymine with two hydrogen bonds and cytosine and guanine with three hydrogen bonds continuously, this process is bi-directional happening at both replication forks simultaneously.
The lagging strand is antiparallel to the leading strand, in addition to having opposite polarities. Primase attaches a RNA primer consisting of around 5-15 nucleotides to the lagging strand in the 5' to 3' direction providing a anchor point to attach DNA polymerase III. DNA polymerase can only synthesise DNA in the 5' to 3' direction, so in order for the lagging strand to be synthesised, DNA polymerase adds complementary nucleotides to the lagging strand, discontinuously, forming segments called okazaki fragments until it reached the next RNA primer, where it breaks off and synthesises another segment. DNA polymerase I now removes the small RNA primer from the lagging strand and synthesises nucleotides to replace the primer. The small gap, referred to as ‘'nicks'', between the DNA is joined up by ligase forming phosphodiester bonds. This process is than repeated until the lagging strand is fully replicated.
Replication terminates at the six termination sequence, which is a recognition site for Tus protein. Tus protein blocks helicase from unwinding the DNA and stops the replication fork from extending. Tus protein allows the replication fork to pass by in one direction but not the other causing the replication fork to be contained in the Tus region which causes the replisome consisting of DNA polymerase III, helicase, primase and ligase to break away ending the replication process.
Another feature of replication in prokaryote replication is the ability for enzymes to correct the errors made during replication. DNA polymerase III has three regions one of which is proof reading site. As the DNA polymerase adds the nucleotides to the template strand one at a time it also checks that the bases are paired correctly. On occasions bases are mismatched (adenine paired with cytosine and guanine paired with thymine), when this occurs DNA polymerase III removes the incorrect nucleotide by breaking the phosphodiester bonds and replaces it with the correct nucleotide. In spite of DNA polymerase having a proof reading site, there are still some nucleotides which are paired incorrectly. Mismatch nucleotides cause the DNA to become distorted allowing the mismatch repair mechanism to remove the errors. The enzyme MutS protein recognises the distortion in the DNA and bind to the distorted area, causing MutH to bind to the GATC site. This signals exonuclease to remove the mismatch nucleotide along with several nucleotides either side of the mismatch. DNA polymerase I now adds the correct nucleotide, ligase joins them together by filling up the ‘'nicks''. Methylase now adds a methyl group to the adenine in the GATC sequence and now the template strand and daughter strand cannot be distinguished.
As we have seen there are many features which are essential for DNA replication in prokaryotes. The replication is semi- conservative which means the new DNA consists of a parent strand and daughter strand, in addition to also being bi- directional. The ability for DNA to repair and maintain errors is a vital feature which minimises mutations. Another essential feature discussed above is the ability to replicate both the leading and lagging strand when they are antiparallel.