Chapter: Pharmaceutical Biotechnology: Fundamentals and Applications - Molecular Biotechnology

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DNA Replication

Although DNA may be differently organized in various organisms one or more double-stranded DNA molecules in a helix conformation are the predominant structures.

 DNA Replication



Although DNA may be differently organized in various organisms one or more double-stranded DNA molecules in a helix conformation are the predominant structures. Strands of DNA are com-posed of four specific building elements (shortly written as A, C, G, and T), the deoxyribonucleotides deoxyadenosine 50-triphosphate, (dATP) deoxy-cytidine 50-triphosphate (dCTP), deoxyguanosine 50-triphosphate (dGTP), and deoxythymidine 50-tri-phosphate (dTTP) linked by phosphodiester bonds. The two strands in the DNA helix are held together through hydrogen bonds between the nucleotides in the various strands. The DNA strands in the helix are complementary in their nucleotide composition: an A in one strand is always facing a T in the other one, while a C is always facing a G (Fig. 5). Moreover, the strands in double-stranded DNA run antiparallel: the 50-P end of the one strand faces the 3 0-OH end of the complementary strand and the other way round.

 

During cell division the genetic information in a parental cell is transferred to the daughter cells by DNA replication. Essential in the very complex DNA replication process is the action of DNA polymerases. During replication each DNA strand is copied into a complementary strand that runs antiparallel. The topological constraint for replication due to the double helix structure of the DNA is solved by unwinding of the helix, catalyzed by the enzyme helicase. In a set of biochemical events deoxyribonu-cleotide monomers are added one by one to the end of a growing DNA strand in a 50 to 30 direction.

 

DNA replication starts from specific sites, called origins of replication (ori). The bacterial chromosome and many plasmids have only one such site. In the much larger eukaryotic genomes there can be hun-dreds of oris present. For circular DNA molecules like bacterial chromosomes and plasmids there are two possible ways for the replication. Semi conservative replication (Fig. 6A) proceeding in the closed circle assuch (Fig. 6B) is one way. The constraint brought forward by the rotation as a consequence of the unwinding (there is no free end!) is resolved by the activity of a special class of enzymes, the topoisome-rases. Alternatively, replication proceeds via a rolling circle model. In that case the replication starts by cutting one of the DNA strands in the ori region and then proceeds as indicated in Figure 6C.

Bacterial plasmids are defined as autonomously replicating DNA molecules. The basis for that state-ment is the presence of an ori site in the plasmids. The qualification autonomous, however, does not imply that a plasmid is independent from host factors for replication and expression. Some plasmids depend on very specific host factors and consequently they can only replicate in specific hosts. Other plasmids are less specific as to their host factor requirements and are able to replicate in a broad set of hosts. As will be demonstrated later, this difference in host range is meaningful when plasmids are exploited in biotechnology.


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