Replication
In their seminal paper on the DNA double helix published in Nature on April 25th, 1953, Watson and
Crick wrote: “It has not escaped our notice that the specific pairing we have postulated immediately
suggests a possible copying mechanism for the genetic material.”
Indeed, during DNA replication within the cell, the two strands of the parent DNA molecule are
separated and on each of them a special enzyme, DNA polymerase, synthesizes the complementary
strands using deoxynucleoside triphosphates (dNTPs) as precursors (see Chemical Structure of Proteins
and Nucleic Acids). The processes of separation of the strands and the synthesis of new strands proceed
in parallel so that the replication fork is formed and it moves along the parent dsDNA molecule. There
are two fundamental facts about all DNA polymerases, which are crucial for the process of DNA
replication:
1 . Since in dNTPs used by the DNA polymerase during synthesis of the new DNA strand the ppp group is
attached to the 5’ end of the nucleoside, the new nucleotide can be added only to the 3’ end of the
growing DNA chain. This fact determines the strict 5’  3’ direction of the extension of the DNA strand.
2. Similarly to RNA polymerase, DNA polymerase requires template for the complementary chain
synthesis but, in contrast to RNA polymerase, DNA polymerase cannot initiate synthesis and requires a
primer. Both DNA and RNA chains can serve as primers, the only requirement is the presence of the 3’
OH group so that DNA polymerase could attach the next nucleotide to it.
The reaction of extension of the primer by DNA polymerase is called the primer extension reaction. This
reaction is crucial for DNA replication and for two major techniques responsible for the current
revolution in biomedicine: PCR and DNA sequencing.
But how can DNA be gradually replicated if the strands are antiparallel while primer extension reaction
can proceed only in the 5’3’ direction?
Replication fork
Well, replication indeed is a process that is far from trivial. The replication fork is shown in the sketch
and the cartoon below. One of the strands, called the leading strand, is synthesized to its (almost) full
length since the direction of synthesis coincides with the 5’3’ direction. The other strand, called the
lagging strand, cannot be synthesized this way. So it is synthesized in the form of very short fragments
(consisting from a hundred nt in case of eukaryotes to a thousand nt in case of prokaryotes), which are
The replication fork. Above: schematics in which
DNA strands are shown in solid lines, RNA primers
are shown in waving lines.
Below: cartoon showing most of players participating
in replication.
known as Okazaki fragments, called after the Japanese molecular biologist who first discovered them
back in 1960s. Unlike in case of PCR and sequencing methods, the cell cannot utilize DNA primers since
DNA polymerase cannot synthesize primers . The cell uses for this purpose the RNA polymerase
enzyme, which does not require a primer (see section Transcription and Open Reading Frame). This
special RNA polymerase, which is not sequence-selective, is called primase (see cartoon above). For DNA
polymerase there is no difference whether the primer is DNA or RNA, it requires only the 3’ OH group to
perform the primer extension reaction.
There are more players in the process of replication, which are shown on the cartoon above. Most
significantly, since DNA strands must be separated at ambient conditions at which the double helix is
very stable, a special molecular motor, called helicase moves along DNA and separates the
complementary strands consuming the ATP energy, of course. Special small proteins, called SSB (for
single-strand-binding), grab strands separated by helicase preventing them from reannealing prior to
synthesis on both of them new strands by DNA polymerase.
Thus, when the replication fork reaches the other end of the molecule, the original strands fully
separate and the two daughter molecules shown at the top of the following cartoon are formed:
Then two additional enzymes enter the scene. One is called DNA polymerase I (DNApol I), which extends
each available 3’ end of Okazaki fragments. In doing so it digest RNA primers ahead of itself since, as
every DNA polymerase, DNApol I has a special domain exhibiting the 5’ exonuclease activity. The rest of
the job, consisting in sealing the gaps between adjacent Okazaki fragments extended by DNApol I, is
fulfilled by yet another enzyme called DNA ligase. As a result both daughter molecules end up being
very similar, as shown at the bottom of the above cartoon. It is essentially the end of the replication
process.
The end problem and its solution
One can ask: but who removes the two 5’-terminal primers, one per each daughter molecule? Of
course, there is no problem with removing the primer. It can be done by a special ribonuclease known
as RNase H, which specifically recognizes DNA/RNA heteroduplex but digests only RNA (that is why it is
called RNase; there are all sorts of nucleases, enzymes digesting nucleic acids: some of them digest only
DNA, some only RNA, others digest both (like the exonuclease domain of DNApol I), some of them digest
inside chain (they are called endonucleases), others only from the end (they are called exonucleases;
some of them like 3’-end others 5’-end)). However, after the removal of the primer there is no enzyme,
which can replace the primer with DNA since no DNA polymerase can extend the 5’ end. This end
problem is a fundamental problem of DNA replication: at each round of replication the double helix
becomes shorted on the size of the primer, which is several dozens bp.
How is the end problem resolved by the cell? Prokaryotes and eukaryotes approach the problem very
differently. Here we encounter one of many points where there these two major kingdoms of living
organisms demonstrate totally different approach.
Prokaryotes
The solution presented by prokaryotes is very elegant, I would say, it demonstrates mathematical
elegance: if there is problems with end, let us get rid of them. Let us make DNA circular: in replicating
the circle no end problem arises since the gap after removal of primer can be filled by DNApol I (actually,
RNase H is also not required since the removal of last primer is performed by DNApol I via its 5’exonuclease activity). ALL prokaryotic (bacterial) DNAs are always circular. It is the case not only for
bacterial genomic DNA but also for plasmids, of course.
Eukaryotes
By whatever reason in eukaryotes genomic DNA molecules are always linear. May be it is because they
are normally much longer than bacterial DNAs. Bacterial genomes consist of several million bp while in
humans we have the whole genome (consisting of 3 billion bp) in the form of 23 chromosomes, each
chromosome carrying exactly one DNA molecule. Therefore one human DNA molecule carries about
100 million bp. DNA molecule of such enormous length, if being circular, could probably encounter
unsurpassable topological problems, such as knotting. Of course, we do not know for sure the reason
but the fact is that ALL eukaryotic genomic DNA molecules are linear.
BOX: The laws of biology
One of many reasons while studying biology is so frustrating for engineers and physicists is the shortage
of solid laws, which could not be violated, similar to, say, conservation laws (conservation of momentum,
angular momentum and energy, for instance). In biology we find that as soon as a specific statement is
made, numerous exceptions immediately pop up. I remember when back in Russia I learned biology going
every winter to 2-week-long school on Molecular Biology near Moscow, the motto of the school was: “the
coyote hunts ONLY at moon nights; but if there no moon, it also hunts if it is hungry”.
Over the years we have learned that there are laws in molecular biology, at least with respect of life as we
know it. Of what we have learned already in the course, we can formulate the following laws, which do not
know exceptions:
1.
In all living organisms genetic information is stored in the form of the DNA double helix
2.
In all prokaryotes, DNA is always circular
3.
In all eukaryotes, genomic (nuclear) DNA is always linear
4.
All proteins synthesized via the ribosome pathway carry only L amino acids when they are exiting
the ribosome
Comments:
To law #3: In addition to DNA in nucleus, the eukaryotic cell also carries DNA in the cytoplasm,
within mitochondria, which is a very short circular dsDNA molecule. This is because, beyond
doubts, mitochondria used to be bacteria, which co-existed with pre-eukaryotic cells living in them
(biologists call such a mutually profitable relationship between different species symbiosis).
Gradually, mitochondria passed almost all their genes to the nucleus. The process stopped only
because at some point the mitochondrial genetic code changed (see pp 67-70 of Unraveling DNA)
To law #4: Via posttranslational changes and via non-ribosome synthesis D amino acids pop up in
the cell (see Background: Chirality)
How is it possible to avoid shortening of linear DNA during the every round of replication? Well, the
truth is that it is exactly what happens in eukaryotes, I mean the shortening. So to protect genes from
being truncated, the chromosomal DNAs carry special buffer regions at their termini, called telomeres.
Telomeres are repeats, many thousand times, of a very simple motif. For all chromosomes in all humans
(actually, in all vertebrates) the repeating sequence is: 5’TTAGGG3’. Mostly it is dsDNA but at the very
end there is a single-stranded 3’ overhang (TTAGGG)n consisting of several dozen repeats.
During individual development of the organism telomeres indeed become shorter and shorter. This
results in the phenomenon of somatic cell mortality: normal somatic cell can sustain only about 50
divisions after which it demonstrates clear signs of senescence and then dies (the phenomenon is
dubbed the Hayflick limit). But what prevents our genomes from complete annihilation over many
generations? The mechanism of telomere extension has been unraveled mostly by the efforts of
Elizabeth Blackburn who was awarded, together with two other scientists, the Nobel Prize in Physiology
or Medicine for 2009. She discovered a special enzyme, which she called telomerase and which extends
telomeres. Since it is impossible to extend 5’-end, telomerase extends the 3’ end instead. It is a good
idea: if the 3’-end is extended significantly, the opposite strand can be synthesized via Okazaki
fragments, similarly to the synthesis on the lagging strand in the replication fork (see above). That is OK
but where is the template?! A sensational discovery Blackburn made consisted in the fact that the
telomerase brings the template with itself: the enzyme is a complex of a protein, reverse transcriptase ,
Telomere extension by telomerase (the enzyme
itself is not shown). The scheme is done for
telomere sequence of single-cell eukaryote
Tatrahymena, which has telemetric repeat TTGGGG.
The telomerase was originally discovered by
Blackburn in Tetrahymena.
which synthesizes DNA on the RNA template, and a pretty long ssRNA molecule (about 500 nt).
Somewhere inside this long RNA there are several telomeric repeats, which serve as the template for
the telomere extension (see the scheme above). The telomeric 3’-overhang serves as primer for reverse
transcriptase.
The discovery of telomerase resolved the mystery of how the replication end problem is resolved for
linear, noncircular DNA molecules of eukaryotes. In normal somatic cells the telomerase gene is
completely shut down: hence the telomere’s shortening with aging and the Hayflick limit. By contrast,
during development of germ cells, in both males and females, the telomerase gene is switched on and
telomerase extends telomeres preparing them for performing their buffer duties in the next generation.
The fact of enormous importance is that in cancer cells the telomerase gene is activated again. This is
why, in contrast to normal somatic cells, cancer cells are immortal: they can divide indefinitely. Most
dramatic example of this immortality is so called HeLa cell line: a cell line based on a sample of cells
taken from a tumor of a female patient in 1951 (her name was Henrietta Lacks, hence HeLa). The cell
line is still in extensive use in many laboratories over the world.