Escherichia coli (E. coli), a bacteria is an example of a prokaryotic organism. For it to grow, reproduce and survive DNA replication is required for cell division. Prokaryotes have two types of nucleic acids, RNA and DNA which are composed of a molecule of a 5 carbon sugar deoxyribose, a nitrogenous base and a phosphate group, collectively known as a nucleotide. In DNA replication, the DNA is first separated into two daughter cells. These new cells carry the same genetic code as the parent cell. DNA replication in most prokaryotes is bidirectional and starts at a single origins of replication (oriC). The oriC structure is 245 base pairs long. There are three 13 (bp) sequences on its left side - the unwinding weak region, due to high levels of adenine and thymine (ATs) nitrogenous bases. The other region of the oriC contains the consensus sequence 5' - GATCTNTTNTTTT - 3'.
The initiation of replication is caused by DnaA, a protein that binds to an area of the origin known as the DnaA box. In E. coli, there are 5 DnaA boxes. Each has a highly conserved four 9 bp (base pairs) consensus sequence 5' - TTATCCACA - 3'. Binding of DnaA to this region allows the helix structure to become unzipped with the aid of DNA helicase (enzyme) by breaking the hydrogen bonds between the complementary base pairs. The uncoiling continues all the way up to an area of the oriC known as DnaB boxes causing it to melt. Melting requires ATP which is hydrolysed by DnaA. The AT bases facilitate melting due to their being held together by two hydrogen bonds unlike the cytosine and guanine (CGs) which are held by three. After melting, DnaA allows helicase (six DnaB proteins) to go to the opposite end of the melted DNA where the formation of replication fork begins, forming the 'Y'-shaped forks which are the actual sites for DNA copying. Before the formation of the forks, the completion of the prepriming structure is vital. Therefore, recruitment of DnaB needs six DnaC proteins, each of which is attached to one subunit of helicase. The replication fork has a continuous synthesis (only requiring one primer) on the leading strand. The continuous synthesis extends the free 3' end by moving towards the replication fork. As for the discontinuous synthesis it moves away from the fork. It requires lots of primers on the lagging strand which is synthesised in short fragments known as Okazaki fragments. These fragments (1000-2000 nucleotides long) are processed by the replication machinery to continuously producing the DNA strands and hence it completes a daughter DNA helix. The Okazaki fragments are covalently joined by a phosphodiester bond with the aid of DNA ligase enzyme.
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Continuation of replication requires that the single-stranded binding proteins (SSBs) to stop the single DNA chains from making any secondary structures, thus blocking them from reannealing. Topoisomerase (DNA gyrase) enzyme causes breaks in the DNA strands and later rejoins them in order to relieve the strain, creating negative supercoils, which are formed by DnaB. The uncoiling of DNA by DnaB helicase permits for DnaG (primase) and RNA polymerase to prime each DNA template so that DNA synthesis can commence. Therefore, DNA replication requires an RNA primer (5-10 nucleotides long) which is synthesized by RNA polymerase complex known as primase, to join RNA nucleotides without needing a pre-existing strand of nucleic acid. The unpaired bases are now free to bind with other nucleotides with the exact complementary bases by covalent bonds with the aid of enzymes called DNA polymerases. They cannot start a new DNA chain from scratch. DNA polymerase I uses its exonuclease activity to digest RNA and replaces it with DNA. DNA polymerase III (the prominent enzyme in prokaryotes) then replaces the primase and attaches DNA nucleotides to the primer only in the 5' to 3' end direction. It is also able to determine the difference between deoxyribonucleotides and ribonucleotides during replication. The phosphodiester linkage is formed when an additional deoxyribonucleoside triphosphate (dNTP) is added to the 3' end of the growing DNA strand. This bond is made by a nucleophillic attack caused by the 3 hydroxyl group on the alpha phosphate of the dNTP. Also, this enzyme must be able to differentiate between correctly and incorrectly paired bases by their shapes, by strictly following the Watson-Crick base pairs rules.
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DNA polymerase I is required for high fidelity copying. If not, then mismatching will occur, forming a bulge. DNA polymerase I (a single polypeptide chain) has three domains: poly activity, proofreading and nickase activity. Its nickase activity can recognises the bulge by using its proofreading activity, causing the enzyme to recognise the bases. To get rid of the bulge the nuclease activity will break the bond concerned which will then diffuse away until the correct base pairing is made. Unpaired bases are removed by DNA glycosylases to produce Apurinic/Apyrimidinic sites - AP sites. DNA polymerase I identifies the nick and removes and replaces a region containing the AP sites. DNA polymerase II breaks away the RNA primer and replaces the RNA nucleotides of the primer with the appropriate DNA nucleotides to fill the nick.
The most critical role of DNA is to serve as a recipe for the production of proteins which include enzymes, structural and functional proteins that comprise of an organism's body and help it to form the critical tasks of staying alive.