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Biology 2e

14.3 Basics of DNA Replication

Biology 2e14.3 Basics of DNA Replication

Learning Objectives

By the end of this section, you will be able to do the following:

  • Explain how the structure of DNA reveals the replication process
  • Describe the Meselson and Stahl experiments

The elucidation of the structure of the double helix provided a hint as to how DNA divides and makes copies of itself. In their 1953 paper, Watson and Crick penned an incredible understatement: "It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material." With specific base pairs, the sequence of one DNA strand can be predicted from its complement. The double-helix 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.

Illustration shows the conservative, semi-conservative, and dispersive models of D N A synthesis. In the conservative model, the D N A is replicated and both newly synthesized strands are paired together. In the semi-conservative model, each newly synthesized strand pairs with a parent strand. In the dispersive model, newly synthesized D N A is interspersed with parent D N A within both D N A strands.
Figure 14.12 The three suggested models of DNA replication. Gray 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 acts 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), which gets incorporated into nitrogenous bases, and eventually into the DNA (Figure 14.13).

Illustration shows an experiment in which E coli was grown initially in media containing superscript 15 baseline upper case N nucleotides. When the D N A was extracted and run in an ultracentrifuge, a band of D N A appeared low in the tube. The culture was next placed in the superscript 14 baseline upper case N medium. After one generation, all of the D N A appeared in the middle of the tube, indicating that the D N A was a mixture of half superscript 14 baseline upper N and half superscript 15 baseline upper N, D N A. After two generations, half of the D N A appeared in the middle of the tube, and half appeared higher up, indicating that half the D N A contained 50% superscript 15 baseline upper N, and half contained superscript 14 baseline upper N only. In subsequent generations, more and more of the D N A appeared in the upper, superscript 14 baseline upper N band.
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. These data support the semi-conservative replication model. (credit: modification of work by Mariana Ruiz Villareal)

The E. coli culture was then placed into medium containing 14N and allowed to grow for several generations. After each of the first few generations, the cells were harvested and the DNA was isolated, then centrifuged at high speeds in an ultracentrifuge. During the centrifugation, the DNA was loaded into a gradient (typically a solution of 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 buoyant density: the density within the gradient at which it floats. DNA grown in 15N will form a band at a higher density position (i.e., farther down the centrifuge tube) 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. And for this reason, therefore, the other two models 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 strands will be complementary to the parental or “old” strands. When two daughter DNA copies are formed, they have the same sequence and are divided equally into the two daughter cells.

Link to Learning

View this video on DNA replication.

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