The process of DNA replication follows a semi-conservative scheme which generates a double helical strand that is composed of one original and one new DNA strand.
The semi-conservative scheme of DNA replication was finalized after several years of analysis of the DNA molecule. In order to solve the topological problem of unwinding the DNA double helix in order for the process of DNA replication to proceed, DNA topoisomerases are needed in order to break and reunite the two strands of the DNA helix. There are three types of DNA topoisomerases that may be involved in DNA replication (Champoux, 2001).Type IA topoisomerases induce a single break in one of the two strands of the DNA molecule and the second DNA strand passes through the gap that is formed. The two broken ends of the DNA strand that was nicked are reunited after the second DNA strand has passed through it. Type IB topoisomerases also follow a similar method as Type IA topoisomerases but the specific details are somewhat different.
Type II isomerases induce two breaks in the DNA double helix which in turn result in a gate through which another part of the DNA molecule can pass through.DNA topoisomerases unwind the DNA strand and counteract the excessive winding that may result if a replication fork is to be created. Thus DNA topoisomerases unzip the double helix in order for the rest of the enzymes to attach to the replication fork. Gyrases are another type of enzymes involved in DNA replication among bacterial central. These enzymes relax the DNA molecule, just like DNA topoisomerases and prevent supercoiling or the tight winding of the helical structure of DNA. DNA replication involves three major phases.
Initiation pertains to the identification of the key sequences on a DNA strand that designates where replication should start. Elongation involves the processes that occur at the replication fork and this is where the new DNA strand is created. Termination involves the completion of the DNA replication. Another key enzyme involved in DNA replication is DNA polymerase which synthesizes new DNA strands in the 5’ ? 3’ direction. DNA polymerases often work as exonucleases which destroy nucleotides and also synthesize new nucleotides as needed.There are two types of exonucleases that work with DNA polymerases.
The 3’ ? to 5’ exonuclease activity is present in both bacterial and eukaryotic DNA polymerases and this capability allows proofreading of the new nucleotides that are integrated into the new DNA strand. The 5’ ? 3’ exonuclease activity of DNA polymerases is not very common but this may be needed in order to remove some part of a DNA strand during discontinuous DNA replication. Research has identified three types of DNA polymerases in bacteria.DNA polymerase I is involved in the replication of the DNA strand, DNA polymerase II is responsible for the repair of DNA regions that are damaged and DNA polymerase III is associated with genome replication (Zhou and Elledge, 2000). DNA polymerases I and II are composed on only one polypeptide chain while DNA polymerase III is a multi-unit protein complex made up of three subunits. DNA polymerase III moves like a sliding clamp that goes through the DNA template during replication.
In eukaryotic cells, there are approximately nine DNA polymerases that are involved in DNA replication.DNA polymerase delta serves as the main enzyme responsible for replication in eukaryotic cells (Gilbert, 2001). In bacterial cells, the enzyme primase is also needed in DNA replication. Primases are RNA polymerases that generates should DNA segments of approximately 4 to 15 nucleotides in length which are added onto the growing DNA strand. These short DNA segments are known as primers onto which the DNA polymerase adds new nucleotides for the elongation stage of DNA replication.
Helicases are another type of enzyme that holds the DNA helix in an unwound configuration in order for the replication process to continue.When the helicase is attached to the DNA molecule, single-strand binding proteins (SSBs) attach to each separated DNA strand and prevent the complementary strands from binding back together. The single-strand binding proteins also stabilize each separated strand of the DNA helix. A specific type of single-strand binding protein in replication protein A (RPA) which identifies that sites along the DNA strand that should be copied first (Bae et al. , 2001). The RPA protein also identifies sites of recombination and repair in the cell.
In eukaryotic cells, the flap endonuclease is responsible for associating DNA polymerase to the 3’ end of an Okazaki fragment. The association facilitates the destruction of the primers that were generated by the enzyme primase. The flap endonuclease is actually a new enzyme that has been discovered to have a cleaning action in the replication fork. It has been suggested that the flap endonuclease cuts the phosphodiester bonds at the attachment point where the relocated region is associated to the primer.
Another suggestion is that instead of employing the flap endonuclease, the enzymes RNase H destroys the RNA portion of the RNA-DNA hybrid. However, this concept should therefore need the presence of a 5’-monophosphate instead of a triphosphate so that flap endonuclease will not have the chance to come into the replication fork configuration. The Tus binding protein is responsible for the detachment of the daughter strand from the replication fork, which then results in the restoration of the double helical DNA configuration, ending the DNA replication process.Telomerases are enzymes that are responsible in adding nucleotide repeats of the sequence (TTAGGG)n at the tips of the chromosomes, also known as telomeres. It has been determined that telomeres shorten as a cell ages and there is a critical length of telomere repeats that determine whether a cell should die.
However, in cancer cells which experience uncontrollable cell division, telomeres are continuously being replicated due to the mutated function of telomerases hence the cell continues to divide resulting in a cancerous tissue or tumor.
