Process of DNA Replication - Coursework Example

DNA replication Introduction DNA is an abbreviation for deoxyribonucleic acid. It is formed by polymerization of nucleotides. Nucleotides are composed of deoxyribose sugar, nitrogen containing base and a phosphate group. Different nucleotides join to one another using phosphodiester bonds between the 5’ carbon on one deoxyribose sugar and the 3’ carbon on the next deoxyribose (Cotteril, 1999). New bases are always added at the 3’ end of the DNA and therefore DNA is synthesized in the 5’ to 3’ direction. There are four different nucleotides which are grouped into two purines (adenine (A) and guanine (G)) and two pyrimidines (cytosine (C) and thymine (T)). The amount of adenine in DNA is equal to the amount of thymine while the amount of cytosine is equivalent to that of guanine in any given DNA (DePamphilis, 1996). This implies that the amount of purines in DNA is equivalent to that of pyrimidines. The structure of DNA was first deduced by Watson and Crick from X-ray diffraction pattern. From these experiments they deduced that DNA is a double stranded helix. Each of these strands is composed of nucleotides which are bonded together covalently (DePamphilis, 2006). The two strands of DNA are linked together by hydrogen bonds which exist between the complementary bases. This hydrogen bonding of bases is referred to as base pairing. During base pairing adenine base pair with thymine while guanine base pair with cytosine. The two strands of DNA are twisted around each other to form a double helix. The backbones of sugar phosphates of the DNA are found on the outer part of the helix while the nitrogenous bases are in the middle of the helix (Vengrova & Dalgaard, 2009). The nitrogenous bases’ outer edges are exposed in the minor and major grooves. Why DNA replication The specific base pairing in the DNA implies that one can be able to work out the sequences in bases of one strand of DNA using another strand. The synthesis of new DNA strands is referred to as DNA replication. Replication of DNA requires the presence of four deoxyribonucleotides (dNTPs). These are: deoxyadenosine triphosphate (dATP), deoxyguanosine triphosphate (dGTP), deoxythymidine triphosphate (dTTP) and deoxycytidine triphosphate (dCTP). Thus dNTPs are required for the synthesis of DNA (Cotteril, 1999). DNA replication is very vital in the process of cell division. Cell division is essential for the production of skin, gastric mucosa, hair and blood. DNA replication usually occurs during the S phase of cell division. DNA replication results in the formation of new chromosomes (Sadava, 2007). DNA synthesis The first synthesis of DNA in trhe test tube was done in 1955 using an extract of Escherechia coli. DNA is synthesized by the DNA polymerase. For the polymerase to synthesize a DNA three components must be available (Sadava, 2007). These are, DNA template from which the polymerase copies the sequence, deoxyribonucleotides (dATP, dCTP, dGTP and dTTP) and a primer strand to which the dNTPs are added. The process of DNA synthesis Three possible ways have been proposed to explain how DNA replicates. These are the conservative replication, semi conservative replication and dispersive replication. The conservative model proposes that DNA is copied to form new strands which form new strand of DNA while leaving the parental DNA strands intact. The semi conservative model proposes that each of the new DNA molecule that is formed is composed of one strand from the parental molecule and another strand from the new daughter DNA. The dispersive model proposes that the new DNA molecules are formed from a mixture of bits from the parental and the daughter molecules of DNA. Key A – Semi conservative model B – Conservative model C – Dispersive model Adapted from: Meselson and Stahl were able to demonstrate experimentally that the DNA replication is semi conservative. In their experiment, they grew E. coli in two different medium, one containing heavy nitrogen (15N) as a source of nitrogen while the other containing normal nitrogen (14N) as a source of nitrogen. After growth, they purified the DNA from the bacteria growing in the two medium and placed the DNA on a cesium chloride gradient (Kornberg & Baker, 2005). From the results, they found that DNA purified from E. coli grown on heavy nitrogen was denser than that grown on normal medium containing normal nitrogen, 14N. meselson and Stahl then grew the bacteria from the medium containing heavy nitrogen to a medium containing normal nitrogen for several generations and placed the DNA on a cesium chloride gradient (Vengrova & Dalgaard, 2009). The F1 generation had DNA with intermediate density between the one from normal nitrogen and the one from the heavy nitrogen (DePamphilis, 1996). The F2 generation bacteria had half of DNA with intermediate density while the other half had density equivalent to that obtained from bacteria grown on medium containing normal nitrogen. None of the DNA obtained had density equivalent to that grown on heavy nitrogen containing medium. From these results, Meselson and Stahl concluded that DNA replication is semi conservative (DePamphilis, 2006). Adapted from: Semi conservative replication of DNA DNA contains two templates which are complementary to one another. Prior to replication, the hydrogen bonds joining the two templates together usually break (Cotteril, 1999). On breakage of these hydrogen bonds, the two strands unwind and separate. The resulting strands are then used as templates for the synthesis of new strands which are complementary to parental strands resulting in the formation of two new strands with each new DNA containing a parental strand and a daughter strand. The synthesis of new DNA strands is catalyzed by DNA polymerase. This involves addition of dNTP toa DNA residues resulting in a DNA strand which is longer by one dNTP and a pyrophosphate (PPi) is released in the process. DNA (n residues) + dNTP DNA (n +1) + PPi DNA polymerase The pyrophosphate which is released is hydrolyzed to form two phosphates. This involves breakage of high energy bond which yields energy that drives the DNA synthesis reaction. The synthesis of new DNA strand occurs in the 5’ to 3’ direction. DNA residues already have bases at the 5’ end which have a free 5’ phosphate. The incoming base is usually attached to the 3’ hydroxyl of the last base. The terminal deoxyribose 3’ hydroxyl is then involved in nucleophilic attack of the innermost alpha phosphate of the incoming dNTP (Kornberg & Baker, 2005). This results in the formation of a phosphodiester bond and liberation of a pyrophosphate and energy which drives the reaction. The new dNTP that is added to the growing DNA strand is usually chosen so that it corresponds to that on the template strand. Thus the nucleotide sequences on the template strand dictates the sequences on the new strand being formed. This results in the formation of DNA that has one strand from the parent DNA and the other being the newly formed strand. This explains the semi conservative replication of DNA. Adapted from: Origin of replication The replication process of DNA begins at the origin of replication. Prokaryotes replication has a single origin of replication (ori) while eukaryotes have many origins of replication. the replication process of DNA occurs in both directions from the origin of replication (DePamphilis, 1996). Thus both DNA strands act as templates for the synthesis of new strands of DNA. During replication, the DNA passes through the replication complex (DePamphilis, 2006). Enzymes necessary for replication The enzymes required for DNA replication includes DNA polymerase, helicases, primases, DNA ligase and telomerase. The helicase enzyme is involved in the unwinding of the double stranded DNA (Cotteril, 1999). The DNA is then kept unwound by the single stranded binding proteins. The primase enzymes are then involved in the synthesis of short fragments of RNA which act as primers for the synthesis of new strands of DNA. In bacteria the DNA polymerase III is involved in the synthesis of the leading and lagging strands of DNA. The polymerase enzyme is kept in place by a clamp protein. In bacteria the DNA polymerase I enzyme is involved in the hydrolysis of RNA primers and replaces them with DNA (Kornberg & Baker, 2005). DNA polymerase is also involved in the proof reading of the newly inserted nucleotides to ensure they are complementary to the template bases. The polymerase enzyme has an exonuclease activity which is involved in cleaving wrongly incorporated bases in the growing strand of DNA (DePamphilis, 1996). The lagging strand of DNA is usually synthesized in fragments referred to as Okazaki fragments since DNA can only be synthesized in the 5’ to 3’ direction. Thus these fragments are joined by the DNA ligase enzymes. DNA topoisomerase is used by bacteria to separate the two circular DNAs that are formed. On the other hand, the telomerase enzyme is used by eukaryotic cells to maintain the chromosomal length. Conclusion DNA replication is an essential process in the life of an organism since it is involved in the cell division process. Replication is usually a semi conservative process which results in formation of new DNA. The newly formed DNA has one strand originating from the parental DNA while the other strand is a daughter strand. The replication process involves synthesis of new strands of DNA and it requires different enzymes. The enzymes required for replication process includes the helicases, the primase, the DNA polymerase, the DNA ligase, telomerase in eukaryotic cells and the topoisomerase in the bacterial cells. Reference Cotteril, S. 1999. Eukaryotic DNA replication: a practical approach. Oxford: Oxford University Press. Kornberg, A. & Baker, T. 2005. DNA replication, Volume 2005, 2nd Ed. London: University Science Books. DePamphilis, M. 1996. DNA replication in eukaryotic cells. London: CSHL Press. DePamphilis, M. 2006. DNA replication and human disease. London: CSHL Press. Vengrova, S. & Dalgaard, J. 2009. DNA replication: methods and protocols. London: Humana Press. Sadava, D. 2007. Life, the science of biology, 8th Ed. New York: Sinauer Associates. Read More