Insight into the mode of DNA replic

Insight into the mode of DNA replication: the Meselson–Stahl experiment
The mode of replication was determined in 1958 by Matthew Meselson and Franklin W. Stahl. They
designed an experiment to distinguish between semiconservative, conservative, and dispersive replication.
First, they needed a way to tell original DNA from newly synthesized DNA. To this end, they grew
Escherichia coliin medium containing
15
N, a heavy isotope of nitrogen. 15
N contains one more neutron than
the naturally occurring
14
N. Unlike radioisotopes, 15
N is stable and is not radioactive. After growing several
generations of bacteria in the
15
N medium, the DNA of E. colibecame denser because the nitrogenous bases
had incorporated the heavy isotope.
The density of the strands was determined using a technique known as density-gradient centrifugation.
A solution of cesium chloride (CsCl) – a heavy metal salt – containing the DNA samples is spun in an
ultracentrifuge at high speed for several hours. Eventually, an equilibrium between centrifugal force and
diffusion occurs, such that a gradient forms with a high concentration of CsCl at the bottom of the tube
and a low concentration at the top. DNA forms a band in the tube at the point where its density is the same
as that of the CsCl. The bands are detected by observing the tubes with ultraviolet light at a wavelength of
260 nm, in which DNA absorbs strongly.
After many generations, Meselson and Stahl transferred the bacteria with heavy (
15N) DNA to a medium
containing only
14
N. What they found was that DNA replicated in the 14
N medium was intermediate in
density between light (
14
N) and heavy (15
N). In the next generation, only DNA of intermediate and light
density was present. The results shown in Fig. 6.2 are consistent only with semiconservative replication. If
replication had been conservative, there would have been two bands at the first generation of replication
– an original
15
N (heavy) double helix and a new 14
N (light) double helix. Additionally, throughout the
experiment, the original DNA would have continued to show up as a
15
N (heavy) band. If the method of
replication had been dispersive, the result would have been various multiple-banded patterns, depending on
the degree of dispersiveness.
Insight into the mode of DNA replication: visualization of replicating bacterial DNA
The semiconservative method of replication was visually verified by J. Cairns in 1963 using the technique
of autoradiography. This technique makes use of the fact that radioactive emissions expose photographic
film. The visible silver grains on the film can then be counted to provide an estimate of the quantity of
radioactive material present. Cairns grew E. coliin a medium containing the base thymine labeled with
tritium, a radioactive isotope of hydrogen (
3
H). The DNA was then extracted from the bacteria and
autoradiographs were made. By analysis of DNA at different time points during replication (it takes
approximately 42 minutes to replicate the entire genome), Cairns showed that replication of the circular
Figure 6.2 (opposite) The Meselson and Stahl experiment to determine the mode of DNA
replication.Meselson and Stahl shifted 15
N-labeled E. coli cells to a
14
N medium for several generations, and then
subjected the bacterial DNA to CsCl gradient ultracentrifugation. The bands after centrifugation come about from
semiconservative replication of
15
N DNA (blue) replicating in a 14
N medium (green). (Inset) (A) Photographs of the
centrifuge tubes under ultraviolet illumination. The dark bands correspond to heavy
15
N-labeled DNA (right) and light
14
N-labeled DNA (left). A band of intermediate density was also observed between these two and is the predominant
band observed at 1.0 and 1.1 generations. This band corresponds to double-stranded DNA molecules in which one
strand is labeled with
15
N, and the other with 14
N. After 1.9 generations, there were approximately equal quantities of
the
15
N/14
N band and the 14
N band. After three or four generations, there was a progressive depletion of the 15
N/14N
band and a corresponding increase in the
14
N band, as expected for semiconservative replication. (B) Densitometer
tracings of the bands in panel (A), which can be used to quantify the amount of DNA in each band. (Reprinted by
permission of Matthew Meselson from: Meselson, M., Stahl, F. 1958. The replication of DNA in Escherichia coli.
Proceedings of the National Academy of Sciences USA44:671–682.)
Chapter 6 112
genome was bidirectional. The two strands in the double helix separate at an origin of replication, exposing
bases to form a cytologically visible replication “eye” or “bubble” that contains two replication forks. The
two replication forks proceed in opposite directions around the circle (Fig. 6.3). During replication, the
chromosome looks like the Greek letter theta (θ) by electron microscopy. Replication intermediates are thus
termed “theta structures.” Cairns’ findings have subsequently been verified by both autoradiographic and
genetic analysis.
6.3DNA synthesis occurs from 5′ to 3′
Before exploring the complexity of eukaryotic DNA replication, some basic principles common to most
DNA replication pathways will be described. We now know that during semiconservative replication, the
new strand of DNA is synthesized from 5′ to 3′. Nucleotides are added one at a time to the 3′ hydroxyl
end of the DNA chain, forming new phosphodiester bonds. Deoxynucleoside 5′ triphosphates (dNTPs) are
the building blocks. The terminal two phosphates are lost in the reaction, making the reaction essentially
irreversible (Fig. 6.4). The choice of nucleotide to add to the chain is determined by complementary base
pairing with the template strand. Details of the exact mechanism for how this process occurs varies for cells,
organelle genomes, plasmids, and viruses. The mode of replication depends, in part, on whether the genome
is circular or linear. The most common mode of replication is semidiscontinuous DNA replication. Other
mechanisms include continuous DNA replication and rolling circle replication.
6.4DNA polymerases are the enzymes that catalyze DNA
synthesis
Enzymes that polymerize nucleotides into a growing strand of DNA are called DNA polymerases. Over the
past few years, the number of known DNA polymerases in both prokaryotes and eukaryotes has grown
tremendously. Bacteria have five different DNA polymerases (Focus box 6.1), whereas mammalian cells are
now known to contain at least 14 distinct DNA polymerases (Table 6.1). In eukaryotes, three different DNA
polymerases are involved in chromosomal DNA replication: DNA polymerase α, DNA polymerase δ, and
DNA polymerase ε. DNA polymerase γis used strictly for mitochondrial DNA (mtDNA) replication. These
four enzymes are referred to as the replicative polymerases, to distinguish them from the remaining polymerases
that are involved in repair processes. The repair polymerases will be discussed in detail in Chapter 7.
All the known DNA polymerases can only add nucleotides in the 5′→3′ direction. In other words, a
DNA polymerase can catalyze the formation of a phosphodiester bond between the first 5′-phosphate group
of a new dNTP and the 3′-hydroxyl group of the last nucleotide in the newly synthesized strand (Fig. 6.4).
But the DNA polymerases cannot act in the opposite orientation to create a phosphodiester bond with the
5′-phosphate of a nucleotide already in the DNA and the 3′-hydroxyl of a new dNTP.
Another feature of DNA polymerases is that they cannot initiate DNA synthesis de novo(Latin for “from
the beginning”). With the exception of DNA polymerase α(the polymerase involved in primer synthesis),
they all require a “primer.” The primer is usually a short RNA chain which must be synthesized on the
DNA template before DNA polymerase can start elongation of a new DNA chain (primers are discussed in
detail below). DNA polymerases recognize and bind to the free 3′-hydroxyl group at the end of the primer.
Once primed, polymerases can extend pre-existing chains rapidly and with high fidelity. Bacterial and
mammalian DNA polymerases can add ~500 and ~50 nt/second, respectively.
6.5Semidiscontinuous DNA replication
The major form of replication that occurs in nuclear DNA (eukaryotes), some viruses (e.g. the papovavirus
SV40), and bacteria is called semidiscontinuous DNA replication. Fundamental features are conserved from
E. colito humans. The differences are in the details; that is, in the specific enzymes and other proteins that
are involved in the process (Table 6.2).