14.3 Basics of DNA Replication

The Science Practice Challenge Questions contain additional test questions for this section that will help you prepare for the AP exam. These questions address the following standards:
[APLO 2.34][APLO 3.3][APLO 4.1]

The elucidation of the structure of the double helix provided a hint as to how DNA divides and makes copies of itself. This model suggests that the two strands of the double helix separate during replication, and each strand serves as a template from which the new complementary strand is copied. What was not clear was how the replication took place. There were three models suggested (Figure 14.12): conservative, semi-conservative, and dispersive.

Figure 14.12 The three suggested models of DNA replication. Grey indicates the original DNA strands, and blue indicates newly synthesized DNA.

In conservative replication, the parental DNA remains together, and the newly formed daughter strands are together. The semi-conservative method suggests that each of the two parental DNA strands act as a template for new DNA to be synthesized; after replication, each double-stranded DNA includes one parental or “old” strand and one “new” strand. In the dispersive model, both copies of DNA have double-stranded segments of parental DNA and newly synthesized DNA interspersed.

Meselson and Stahl were interested in understanding how DNA replicates. They grew E. coli for several generations in a medium containing a “heavy” isotope of nitrogen (¹⁵N) that gets incorporated into nitrogenous bases, and eventually into the DNA (Figure 14.13).

Figure 14.13 Meselson and Stahl experimented with E. coli grown first in heavy nitrogen (¹⁵N) then in ¹⁴N. DNA grown in ¹⁵N (red band) is heavier than DNA grown in ¹⁴N (orange band), and sediments to a lower level in cesium chloride solution in an ultracentrifuge. When DNA grown in ¹⁵N is switched to media containing ¹⁴N, after one round of cell division the DNA sediments halfway between the ¹⁵N and ¹⁴N levels, indicating that it now contains fifty percent ¹⁴N. In subsequent cell divisions, an increasing amount of DNA contains ¹⁴N only. This data supports the semi-conservative replication model. (credit: modification of work by Mariana Ruiz Villareal)

The E. coli culture was then shifted into medium containing ¹⁴N and allowed to grow for one generation. The cells were harvested and the DNA was isolated. The DNA was centrifuged at high speeds in an ultracentrifuge. Some cells were allowed to grow for one more life cycle in ¹⁴N and spun again. During the density gradient centrifugation, the DNA is loaded into a gradient (typically a salt such as cesium chloride or sucrose) and spun at high speeds of 50,000 to 60,000 rpm. Under these circumstances, the DNA will form a band according to its density in the gradient. DNA grown in ¹⁵N will band at a higher density position than that grown in ¹⁴N. Meselson and Stahl noted that after one generation of growth in ¹⁴N after they had been shifted from ¹⁵N, the single band observed was intermediate in position in between DNA of cells grown exclusively in ¹⁵N and ¹⁴N. This suggested either a semi-conservative or dispersive mode of replication. The DNA harvested from cells grown for two generations in ¹⁴N formed two bands: one DNA band was at the intermediate position between ¹⁵N and ¹⁴N, and the other corresponded to the band of ¹⁴N DNA. These results could only be explained if DNA replicates in a semi-conservative manner. Therefore, the other two modes were ruled out.

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. When two daughter DNA copies are formed, they have the same sequence and are divided equally into the two daughter cells.