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DNA molecules are unbelievably long. One mammalian chromosome DNA molecule may be about 1 meter long when unwound. However, it has to reside in a nucleus 5-6 orders of magnitude smaller. This causes it to be folded up in chromosomes in a highly organized fashion4.
A single strand of DNA is a linear polymer that consists of four different building units termed the nucleotides. All the genetic information is located in chromosomes found in the nucleotides that form the polymers. Each nucleotide is composed of: a nitrogenous base known as purine or pyrimidine. Purine can either be Adenine (A) or guanine (G) while pyrimidine can be either Cytosine (C) or Thymine (T); a sugar, deoxyribose; and a phosphate group4. The type of the nitrogenous a nucleotide possesses determines the identity of that particular. A single DNA strand can accommodate hundreds of millions of nucleotides. Typically, T forms hydrogen bonds with A while C forms hydrogen bonds with G. Eukaryotic cell chromosomes have two of these strands winding together to form a double-helix DNA molecule. The base pairs in the DNA strands act as information manuals, but the actual translation of the information to decode cellular action is undertaken by the Ribonucleic Acid, RNA.
Much scientific work has gone into trying to understand how deoxyribonucleic acid (DNA) replicates itself. DNA replication is a multiplication process involving the use of existing DNA strand as the template to synthesis new, identical strand. In deed, the founder of this process, Arthur Kornberg (1960), compared it to the recording of instructions for performing a particular task in a tape4. Copies of this tape, with the exact information, can be made so that this information can be used again else where.
However, the reality of the process of replication is far too complicated than this simple comparison. Most researches to study the process of DNA replication are done using simple Bacterial Cells. These studies have unfortunately not been able to establish all the answers, especially where replication of Eukaryotic Cells are concerned. Nonetheless, the studies have been able to establish basic steps of replication that are generally agreed upon by scientists, who continue to rely on this information as the basis for farther studies and experimentations5.
These in-vitro syntheses make use of coded/labeled extracts of, for instance, E. coli DNA replication genes. The conversion of, for example X174 single-stranded DNA to double-stranded E. coli DNA requires extracts of E. coli replication genes.
In the same line, replication of Eukaryotic DNA involves the use of the individual cell’s existing DNA strands as template. Each of the two strands of the Eukaryotic DNA acts as a template for new DNA strands5. One cycle of replication therefore results in two new strands that interact with the templates to form Duplex DNA strands.
This paper will therefore be looking to establish a comparison between the processes of replication in-vivo and in-vitro, what is different, and what is similar about the two. This comparison may perhaps be best brought out by molecular analysis of what processes are involved, and the role of every individual enzyme in both cases.
In-vivo Replication of DNA
The process of DNA replication, whether in the genome of bacteria having up to 4 million or common House Mouse genome with up to 3 billion2, involves extremely sophisticated and coordinated cascade of molecular events that can be divided into initiation; unwinding; synthesis of primer; and elongation stages.
To facilitate initiation, the initiator proteins bind at the point of origin of replication, a base pair termed the oriC2. The binding sets off a series of events that result in the unwinding of the double-helical strands of DNA into two single-stranded molecules. For the two strands to unwind, the hydrogen bonds between complementary base pairs must be broken down. This has been shown to be the role of DNA helicase enzymes. To prevent the two unwound strands from rejoining, single-strand-binding proteins keep them stable until the process of elongation begins. The origin of replication is the point where a DNA strand starts to unwind into single strands. Replication usually precedes the unwinding, occurring simultaneously on the two single strands but in opposite directions. As such, two replication folks that move along the DNA strands as they replicate are formed4.
However, the actual synthesis of the DNA molecule starts at primer synthesis stage. Primers are usually short nucleotides that are synthesized by RNA polymerase enzymes called Primase. Primers are important because they are capable of synthesizing Ribonuleotides, as opposed to the DNA polymerases which are only capable of adding oxyribonucleotides to the 3’-OH group of already existing DNA strands and are incapable of actual synthesis5. After the elongation process is completed, the primers are removed and replaced by DNA nucleotides5.
The process of elongation involves adding of nucleotides to the DNA strand immediately after primer formation. The sequence of addition of nucleotides to the new DNA strand is dictated by the template strand. This process is almost entirely the role of DNA polymerase, which moves only in one direction, from the 3’ end of the new strand, adding nucleotides to elongate the strand5.
In-vitro Synthesis of DNA
The model for this in-vitro synthesis will be based on the 1973 study by Bertold Francke and Tony Hunter to investigate the in-vitro synthesis of DNA by use of polyoma-infected cells. This study investigated the function of polyoma ts-a using a DNA-synthesizing system. The study shows the constant requirement of early mutants of polyoma for the synthesis of viral DNA. The ts-a function seems to be necessary for the initiation of new rounds of viral DNA replication3.
Viral DNA synthesis usually occurs on pre-existing intermediates of replication4. Therefore, the steps involved in this study dwells more on the elongation of chains during every round of DNA replication.
To portray the effect of temperature on this process, the cells were either maintained at 32 or shifted to 39 for 2 hours before preparing the synthesizing system3. In the case of ts25 (ts-a group) infection, it was clear that the shift to 39 had a drastic effect of reducing in-vitro incorporation into viral DNA fraction. This fact was confirmed by the analysis of sucrose gradient of the in-vitro product. The results showed that the cell enzymes, lysate, from the cells maintained at 32 gave rise to 20S viral DNA. On the other hand, the viral DNA fraction from lysate of the cells shifted to 39 showed radioactivity only in very low-molecular-weight DNA with no evidence of 20S viral DNA3. However, lysates from both shifted and non-shifted ts1260-infected cells showed good incorporation into 20S viral DNA. There seemed to be no evidence of a shut-off in viral DNA synthesis in-vitro when lysates from ts25-infected cells maintained at 32 were incubated at 39 3. The extent of cellular DNA synthesis in lysates from ts25-infected cells was the same whether or not a shift to the non-permissive temperature had been made. This corresponds to the situation that occur in-vivo. Analysis of the in-vitro product synthesized at 32 by ts25- and ts1260-infected cell lysates showed that approximately half (50% and 60%, respectively) was mature form I viral DNA3.
The better part of the replication cycle of polyoma DNA is carried out by cellular functions, with a virus-specific event required only for the initiation step. For this reason, one would expect the temperature dependence of DNA chain elongation to be the same in ts25- and ts1260-infected cell lysates. This fact is confirmed by: First, subjecting the initial DNA synthesis in ts25- and ts1260-infected cell lysates to four different temperature spectra. This established the activation energy of both viral and cellular DNA synthesis. The activation energies for viral DNA synthesis with both ts25 and ts1260 and for cellular DNA synthesis in both cases were all very similar. This finding seems to suggest that the mutation of ts-a function does not alter the activation energy for the elongation DNA strands. Second, viral DNA synthesis has been shown to occur at least partly in a discontinuous fashion by a process which is thought to reflect the general mechanism of DNA replication in eukaryotic cells.
The synthesis of DNA in prokaryotic cell genomes can be initiated by RNA primers. The RNA primers are subsequently elongated by DNA polymerases except in viral genomes. The replication process therefore involves DNA chains with RNA primer chains at their 5'-ends, which are held to the template DNA strand by hydrogen bonds4. The primer RNA chain is attached to the new DNA strand by the forces of 3':5'-phosphodiester bond, which can effectively be interrupted by ribonuclease H. however, the participation of ribonuclease H in in-vivo synthesis of RNA-primed DNA is still subject to discussion5.
Breakthroughs have been made in the study of the process of replication as the four stages involved are currently understood in their most basic form. However, more study should still be conducted to be able to understand how errors in this process of replication contribute to diseases.