Semiconservative Replication Of DNA

Living organisms perpetuate their kind through reproduction. This may be simple duplication (cell
fission) as a bacteria or complex modes of sexual reproduction as in higher plants and animal. In all
cases, however, reproduction entails the faithful transmission of the genetic information of the
parents to the progeny. Since the genetic information is stored in DNA, the replication of DNA is
central to all of biology.

When Watson and Crick proposed the double helix structure of dna with its complementary base-
pairing, they immediately recognized that the base-pairing specificity could provide the basis for a
simple (superficially simple, at least) mechanism for dna duplication. If the two complementary
strands of a double helix separated (by breaking the hydrogen bonds of each base-part), each
parental strand could direct the synthesis of a new complementary strands because of the specific
base-pairing requirements (Fig. 5.12). That is, each parental strand. Adenine, for example, in the
parent strand would serve as a template via its hydrogen-bonding potential for the incorporation of
thymine in the nascent complementary strand. This mechanism of DNA replication is called
semiconservative replication, since each of the complementary strands of the parental double helix
is conserved (or the double helix is “half-conserved”) during the process.

In considering possible mechanisms of DNA replication, three different hypothetical modes are
apparent (fig. 5.13). in addition to (1) semiconservative replication, one can propose(2)
“conservative” replication in which the parental double helix remains intact(is totally conserved) and
somehow directs the synthesis of a “progeny” double helix composed of two newly synthesized
strands, and (3) “dispersive” replication in which segments of parental strands and progeny o
nascent strands become interspersed through some kind of a fragmentation, synthesis, and rejoining
process.

The “Meselson-Stahl experiment”

The results of the first critical test of Watson and Crick’s proposal that dna replicates
semiconservatively were published in 1958 by M.S. Meselson and F.W. Stahl. Their results showed
that the chromosome (now known to contain a single Watson-Click double helix of dna)of the
common colon bacillus Escherechia coli replicated semiconservatively.

Meselson and Stahl grew E. Coli cells for many generations in a medium in which the heavy isotope
of nitrogen , 15N, had been substituted for the normal, light isotope, 14N. The purine and pyrimidine
bases in DNA contain nitrogen thus, the DNA of cells grown on medium containing 15N will have a
greater density (weight per unite volume) than the DNA of cells grown on medium containing 14N.
Since molecules of different densities can be separated by a procedure called equilibrium density
gradient centrifugation , Meselson and Stahl were able to distinguish between the three possible
modes of DNA replication by following the changes in the density of DNA of cells grown on 15N
medium and then transferred to 14N medium for various periods of time (so- called density transfer
experiments).

The density of most DNAs is about the same as the density of concentrated solutions of heavy salts
such as cesium chloride (CsCl). For example, the density of 6 M CsCl is about 1.7 g/cm3. Escbericbia
cole DNA containing 14N has a density of E. coli Dna to 1.724 g/cm3.

When a heavy salt solution such as 6M CsCl is centrifuged at very high speeds (30,000-50,000
revolutions per minute) for 48-72hours, an equilibrium-density gradient is formed (fig. 5.14). the
centrifugal force caused by spinning the solution at high speeds sediments the salt toward the
bottom of the tube. Diffusion, on the other hand result in movement of salt molecules back toward
the top ( low salt concentration) of the rube. After a sifficient period of high speed centrifugation, an
equilibrium between sedimentation and difusion is reached, at which time a linear gradient of
increasing density axists from the top of the tube to the bottom of the tube (fig. 5.14). If DNA is
present in such a gradient, it will move to a position where the density of the salt solution is equal to
its own density. Thus, if a mixture of E. Cooli DNA containing 14N (“heavy” DNA) and E. Coli DNA
containing 14N (“light” DNA) is subjected to CsCl equilibrium density gradient centrifugation , the
DNA molecules will separate into two “bands”, one containing “heavy” DNA and one containing
“light” DNA (Fig. 5.14).

Meselson and Stahl took cells that had been growing in medium containing 15N for several
generations (and thus contained “heavy” DNA), washes them to remove the 15N – containing
medium, and transferred them to medium containing 14N. After allowing the cells to grow in the
presence 14N for varying periods of time, the DNA was extracted and analyzed in CsCl equilibrium
density gradients. The result of their experiment (Fig. 5.15) are only consistent with
semiconservative replication, excluding both conservative and dispersive medels of DNA synthesis.
All the DNA isolated from cells after one generation of growth in medium containing 14N had a
density halfway between the densities of “heavy” DNA and “light” DNA. This intermediate density is
usually referred to as “hybrid” density. After two generation of growth in medium containing 14N,
half of the DNA was of “hybrid” density and half was “light”. These results are precisely those
predicted by the Wtson and Crick semiconservative mode of replication (Fig. 5.15). One generation
of semiconservative replication of a parental double helix containing 15N in medium containing only
14
N would produce two progency double helices both of which had 15N in one strand (the “old”
strand) and 14N in the other strand (the “new” strand). Such molecules would be of “hybrid” density.

Conservative replication would not produce any DNA molecules with “hybrid” density, after one
generation of conservative replication of “heavy” DNA in “light” medium, half of the DNA would still
be “heavy” and the other half would be “light”. If replication were dispersive, Meselson and Stahl
would have observed a shift of the DNA from “heavy” toward “light” in each generation (i.e, “half
heavy” or “hybrid” after one generation, “quarter heavy” after two generation, etc). Meselson and
Stahl’s results are clearly inconsistent with either of these possibilities.

Subsequent studies have verified Meselson and Stahl’s conclusion that DNA replication is
semiconservative and have extended it to many other organism, including higher plants and animals.