DNA synthesis requires a primer usually made of RNA. A primase synthesizes the ribonucleotide primer ranging from 4 to 12 nucleotides in length. DNA polymerase then incorporates a dNMP onto the 3' end of the primer initiating leading strand synthesis. Only one primer is required for the initiation and propagation of leading strand synthesis.
2. DNA polymerase then incorporates a dNMP onto the 3" end of the primer and initiates lagging strand synthesis. The polymerase extends the primer for about 1,000 nucleotides until it comes in contact with the 5' end of the preceding primer. These short segments of RNA/DNA are known as Okazaki fragments.
Phosphoramidites that allow the generation of oligonucleotides containing site-specific lesions have been vital components for studying the mechanism of DNA repair. New DNA lesions are still being discovered and the study of their biological consequences will require their site-specific incorporation into oligonucleotides. The authors conclude that the increased availability of phosphoramidites for the synthesis of lesion-containing oligonucleotides should facilitate many future discoveries in the broad area of DNA damage and repair.
The authors note that the detailed studies of the molecular mechanisms of DNA repair pathways were made possible by using site-specifically modified oligonucleotides and that the availability of phosphoramidites to synthesize oligonucleotides with DNA lesions has contributed to the field. They illustrate the article using primarily structural studies in the following examples:
5. The 3' hydroxyl group on the 3' nucleotide terminus is then covalently joined, using DNA ligase, to the free 5' phosphate of the previously made lagging segment.
The introduction of methods for the chemical synthesis of oligo- and poly- deoxyribonucleotides (DNA sequences) has had a very considerable effect on the development of molecular biology. This is clearly apparent from other sections of the topic. The three most important factors to be taken into account in the chemical synthesis of DNA sequences are:(1) the choice of suitable protecting groups for the 2′-deoxyribonucleoside building blocks , (2) the development of phosphorylation procedures that are suitable for the introduction of the internucleotide linkages, and (3) the purification of the synthetic DNA sequences themselves. The choice of protecting groups is of crucial importance. The protecting groups selected should be easy to introduce; they should also remain completely intact throughout the assembly of the DNA sequences and be easily removable under conditions where the synthetic DNA is completely stable.
An enzyme, DNA polymerase, is required for the covalent joining of the incoming nucleotide to the primer. To actually initiate and sustain DNA replication requires many other proteins and enzymes which assemble into a large complex called a replisome. It is thought that the DNA is spooled through the replisome and replicated as it passes through.
The major catalytic step of DNA synthesis is shown below. Notice that DNA synthesis always occurs in a 5' to 3' direction and that the incoming nucleotide first base pairs with the template and is then linked to the nucleotide on the primer.
There are essentially three phosphorylation methods (Fig. 1) that have been used successfully in the chemical synthesis of relatively high molecular weight DNA sequences.
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Since all known DNA polymerases can synthesize only in a 5' to 3' direction a problem arises in trying to replicate the two strands of DNA at the fork.
Notice that the top strand must be discontinuously replicated in short stretches thus the replication of both parental strands is a semidiscontinuous process. The strand that is continuously synthesized is called the leading strand while the strand that is discontinuously synthesized is called the lagging strand.
Relatively high molecular weight DNA sequences have been prepared successfully by the phosphotriester approach in solution by following essentially the procedure indicated in outline in Figure 1a. However, solution-phase synthesis is relatively laborious in that chromatographic purification steps are usually necessary after each coupling step. Nevertheless, if a very large quantity of a specific sequence is required (see text below), solution-phase synthesis may very well prove to be the method of choice. If, on the other hand, relatively small (i.e., milligram to gram) quantities of material are required for biological or biophysical studies, there is little doubt that solid-phase synthesis is to be preferred. While all three of the above phosphorylation methods (Fig. 1) have been used in solid-phase synthesis, the phosphoramidite approach (9) has emerged as the method of choice. This is mainly because its use leads to high coupling efficiencies and no significant side reactions. Furthermore, most commercial automatic synthesizers have been designed specifically to accommodate phosphoramidite chemistry. The main advantages of solid-phase synthesis, particularly by the phosphoramidite approach, are: (1) that it is very rapid and a DNA sequence containing, say, 50 nucleotide residues can easily be assembled and unblocked within one day; (2) only one purification step is required at the end of a synthesis as the growing DNA sequence is attached to a solid support (such as controlled pore glass [CPG] or polystyrene), and the excesses of all reagents are washed away; (3) all chemical reactions can be made to proceed in very high yield by using large excesses of reagents; and (4) the whole process may be fully automated in a DNA synthesizer. Solid-phase DNA synthesis has been developed to such an extent that the whole process can be carried out by a competent technician with no specialist knowledge of nucleotide chemistry. Automatic synthesizers, some of which are capable of assembling several different specific DNA sequences simultaneously, are readily available, and all the necessary building blocks [particularly phosphoramidites 17] and other reagents and solvents may be purchased in containers that are designed to be attached directly to the synthesizer.
In this review article, Young K. Cheun & Orlando D. Schärer of the Center for Genomic Integrity, Institute for Basic Science, Ulsan National Institute of Science and Technology discuss why research into DNA damage and repair is facilitated by the availability of monomers containing ‘damaged’ bases.