Topics in Nanobiotechnology
Verónica Saravia
OKAZAKI FRAGMENTS
How they were discovered
After the discovery of DNA polymerase by Arthur Kornberg, the properties of the enzyme became quite well
known. One of the most critical is that DNA polymerase (in fact all known DNA polymerases) synthesize DNA in
one direction only: 5' to 3'. This fact led to a dilemma regarding how the semiconservative model would work for a
DNA molecule. Reiji Okazaki was a brilliant Japanese experimenter who took on this problem. Okazaki reasoned
that there were three possibilities for replicating a double-stranded DNA molecule: continuous, semidiscontinuous
or discontinuous. The continuous model is impossible, based upon the nature of DNA polymerases (replication in
only one direction). Therefore he needed to demonstrate that one of the other two models was actually taking place.
Any discontinuous synthesis requires that there be, at least transiently, small pieces of DNA in a replicating
structure. Okazaki decided to look for these small pieces. He employed the ultracentrifuge to do this. The separation
was based upon size, so that he could see these smaller pieces and also follow what happens to them during
replication. In order to follow the course of DNA replication, Okazaki and his colleagues exposed the replicating
DNA to short pulses (about five seconds) of tritiated radioactive nucleotides, followed by the addition of an
excess of normal cold (nonradioactive) nucleotides. This sort of pulse-chase experiment resulted in label being
present only in the DNA that was synthesized during the short period of the pulse. Soon after the pulse, they isolated
the DNA and separated the individual strands from one another in alkaline solution. The various pieces of DNA
could then be sorted out by size: the alkaline solution of DNA was placed on a “sucrose gradient” and spun in an
ultracentrifuge. The bigger pieces of DNA settled more rapidly in such a sedimentation velocity experiment as this
(the sucrose served to stabilize the resulting separations until the investigator could look at them). The scientists then
looked for the presence of label on the spun pieces of DNA. Label occurred on two sizes, one very long, and the
other only on small fragments of 1000 to 2000 nucleotides in length.
Were the smaller fragments artificially induced breakdown products of normally larger pieces? No: when Okazaki
extended the length of the exposure pulse to 30 seconds, a far greater fraction of the total label ended up in long
DNA strands. A similar result was obtained if the period of “cold chase” was prolonged prior to isolation of the
DNA. Clearly the fragments existed as such only temporarily, and soon became incorporated into the growing DNA
strands.
As it turns out, normal 5 3 polymerases are responsible for the synthesis of these Okazaki fragments. Isolation of the
fragments and digestion with 3 exonuclease revealed that the label was added at the 3 end of the fragments, as would
be expected if the DNA fragments were synthesized by poly-III or another polymerase adding bases at the free 3 –
OH end. Finally, the fragments were joined into DNA strands by a DNA ligase enzyme, and mutants that were
ligase-negative (lack a functional ligase) failed to show the pulse-chase assembled into larger fragments.
Okazaki concluded that DNA replication proceeds by a discontinuous mechanism. His data actually suggested that
both strands are copied discontinuously. It wasn't until he used a mutant deficient in a particular repair process
(uracil excision) that he understood that fragments of one strand produced by this repair had nothing to do with the
actual replication process. The small fragments that can be observed during the short radiolabeling periods are called
Okazaki fragments in his honor.
DNA replication: lagging strand
As we know today the process by which DNA is copied is semiconservative, which is to say that after replication
each of the daughter double helices contains one old strand and one new one. Polymerization of new DNA chains is
catalyzed by an enzyme called DNA polymerase, and the substrates for DNA synthesis are deoxyribonucleoside 5’triphosphates (dNTPs). During replication, small segments of the double helix are opened to form regions of
single-stranded DNA. These regions serve as templates for the synthesis of new complementary strands. As the
double helix opens, substrate nucleotides pair with the template (A pairing with T and G with C) and are thereby
selected for incorporation into the growing strand of DNA. DNA polymerase moves along the template and
catalyzes addition of the incoming substrate nucleotides to the 3’ end of the growing DNA chain. Because the two
strands of the template DNA are antiparallel, half the daughter strands must grow in the 5’ to 3’ direction and half in
the 3’ to 5’ direction. But in fact, all known DNA polymerases synthesize DNA in the same direction, with chain
growth proceeding in the 5’ to 3’ direction. Those strands that appear to be growing in the 3’ to 5’ direction are
actually synthesized as short fragments by DNA polymerases that are travelling away from. How then are they
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Topics in Nanobiotechnology
Verónica Saravia
started? All DNA chains are initiated by short pieces of RNA called primers which are synthesised by an enzyme
called DNA primase. Synthesis of the leading strand at each replication fork is continuous and therefore requires
only one primer. In contrast, synthesis of the lagging strand requires a new RNA primer for each Okazaki fragment.
Synthesis of an Okazaki fragment requires five steps. First DNA primase synthesise a short RNA primer. DNA
polymerase then takes over to synthesize the main body of the Okazaki fragment. Subsequently the RNA primer is
removed by an RNase that recognizes the RNA component of DNA:RNA duplexes. The gap produced by removal
of the primer is then filled in by a separate DNA polymerase (a repair polymerase) using the 3’ end of the adjacent
Okazaki fragment as its primer. At this point, all of the template nucleotides are paired with bases on the newly
formed strand, but there is a break in the phosphodiester backbone. The break is sealed by an enzyme called DNA
ligase.
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