11.2 Dna replication  (Page 16/12)

Learning objectives

• Explain the meaning of semiconservative DNA replication
• Explain why DNA replication is bidirectional and includes both a leading and lagging strand
• Explain why Okazaki fragments are formed
• Describe the process of DNA replication and the functions of the enzymes involved
• Identify the differences between DNA replication in bacteria and eukaryotes
• Explain the process of rolling circle replication

The elucidation of the structure of the double helix by James Watson and Francis Crick in 1953 provided a hint as to how DNA is copied during the process of replication . Separating the strands of the double helix would provide two templates for the synthesis of new complementary strands, but exactly how new DNA molecules were constructed was still unclear. In one model, semiconservative replication , the two strands of the double helix separate during DNA replication, and each strand serves as a template from which the new complementary strand is copied; after replication, each double-stranded DNA includes one parental or “old” strand and one “new” strand. There were two competing models also suggested: conservative and dispersive, which are shown in [link] .

Matthew Meselson (1930–) and Franklin Stahl (1929–) devised an experiment in 1958 to test which of these models correctly represents DNA replication ( [link] ). They grew E. coli for several generations in a medium containing a “heavy” isotope of nitrogen ( ¹⁵ N) that was incorporated into nitrogenous bases and, eventually, into the DNA. This labeled the parental DNA. The E. coli culture was then shifted into a medium containing ¹⁴ N and allowed to grow for one generation. The cells were harvested and the DNA was isolated. The DNA was separated by ultracentrifugation, during which the DNA formed bands according to its density. DNA grown in ¹⁵ N would be expected to form a band at a higher density position than that grown in ¹⁴ N. Meselson and Stahl noted that after one generation of growth in ¹⁴ N, the single band observed was intermediate in position in between DNA of cells grown exclusively in ¹⁵ N or ¹⁴ N. This suggested either a semiconservative or dispersive mode of replication. Some cells were allowed to grow for one more generation in ¹⁴ N and spun again. The DNA harvested from cells grown for two generations in ¹⁴ N formed two bands: one DNA band was at the intermediate position between ¹⁵ N and ¹⁴ N, and the other corresponded to the band of ¹⁴ N DNA. These results could only be explained if DNA replicates in a semiconservative manner. Therefore, the other two models were ruled out. As a result of this experiment, we now know that during DNA replication, each of the two strands that make up the double helix serves as a template from which new strands are copied. The new strand will be complementary to the parental or “old” strand. The resulting DNA molecules have the same sequence and are divided equally into the two daughter cells.