ZOOHCC - 501: Molecular Biology (Theory)
Unit 2: DNA Replication
Molecular Mechanism of Bacterial DNA Replication
A typical bacterial cell has approximately 1-4 million base pairs of
DNA, compared to the 3 billion base pairs of the common house mouse (Mus
musculus) genome. However, even in bacteria with small genomes, DNA
replication involves an incredibly sophisticated and highly coordinated
series of molecular events. These events are divided into four main stages:
initiation, completion, primer synthesis, and elongation.
Initiation and Unwinding
During initiation, a so-called initiator protein binds to an origin of
replication, a base-paired sequence of nucleotides known as oriC. This
binding triggers an event that unwinds the DNA double helix into two
single-stranded DNA molecules. Several protein groups are involved in this
process (Figure 1). For example, DNA helicases serve to break the hydrogen
bonds that bind complementary nucleotide bases. These hydrogen bonds are key
features of James Watson and Francis Crick's three-dimensional DNA models.
As newly unwound chains tend to recombine, another group of proteins called
chain binding proteins keep them stable until elongation begins. A third
family of proteins, topoisomerases, relieves some of the torsional stress
caused by double helix unwinding.
As mentioned earlier, the place where a strand of DNA begins to wrap around
two separate single strands is called the origin of replication. when the
double helix is unwound, replication proceeds along his two single strands
simultaneously, but in opposite directions (i.e., left to right on one
strand). , from right to left on the other strand). This creates two
replication forks that replicate as they move along the DNA.
Primer Synthesis
Primer synthesis marks the initiation of the actual synthesis of new DNA
molecules. Primers are short nucleotides (about 10-12 bases in length)
synthesized by an RNA polymerase enzyme called primase. A primer is required
because it is the enzyme that actually adds nucleotides to new DNA strands.
DNA polymerases can only add deoxyribonucleotides to the 3'-OH groups of
existing strands, making synthesis de novo. because it cannot start.
Primase, on the other hand, can add ribonucleotides de novo. Then, after
extension is complete, the primer is removed and replaced with DNA
nucleotides.
Elongation
Finally, elongation (adding nucleotides to the new DNA strand) begins after
the primer is added. Synthesis of the growing strand involves adding
nucleotides one at a time in the exact order dictated by the original
(template) strand. One of the key features of the Watson-Crick DNA model is
that adenine is always paired with thymine and cytosine is always paired
with guanine. For example, if the original strand is A-G-C-T, the new strand
is T-C-G-A.
DNA is always synthesized in the 5' to 3' direction. That is, nucleotides
are added only to the 3' end of the growing strand. As shown in Figure 2,
the 5' phosphate group of the new nucleotide bonds to the 3' OH group of the
last nucleotide of the growing strand. Scientists have yet to identify a
polymerase that can add bases to the 5' ends of DNA strands.
DNA polymerase only moves in one direction
After the primer is synthesized on the DNA strand and the DNA strand is
unwound, synthesis and extension proceed in only one direction. As mentioned
above, the DNA polymerase can only add to her 3′ end, so her 5′ end of the
primer remains unchanged. As a result, synthesis is directly carried out
only along the so-called leading strand. This instantaneous replication is
called continuous replication. The other strand (5′ to her from the primer)
is called the lagging strand, and replication along it is called
discontinuous replication. The duplex must relax to some extent before
initiating synthesis of another primer further up the lagging strand.
Synthesis can then proceed from her 3' end of this new primer. Then the
double helix unwinds some more, followed by another surge of replication. As
a result, replication along the lagging strand only occurs in short,
discontinuous bursts
The newly synthesized fragments of DNA along the trailing strand are called
Okazaki fragments, named after their discoverer, the Japanese molecular
biologist Reiji Okazaki. Okazaki and his colleagues made their findings by
performing so-called pulse-chase experiments, in which replicating DNA was
exposed to short 'pulses' of isotope-labeled nucleotides and the length of
time the cells were exposed to unlabeled nucleotides was varied. I did. This
later stage is called ``chasing'' (Okazaki et al., 1968). The labeled
nucleotide was incorporated into the growing DNA molecule only during the
first few seconds of the pulse. Afterwards, only unlabeled nucleotides were
incorporated during the chase. The scientist then centrifuged the newly
synthesized DNA and observed that the shorter the trace, the more
radioactivity appeared in the "slow" DNA. Sedimentation velocity was
determined by size. Small fragments settled more slowly than large ones due
to their lighter weight. When the researchers increased the length of the
chase, the radioactivity of the 'fast' DNA increased, with little or no
increase in the radioactivity of the slow DNA. The researchers correctly
interpreted these observations to mean that the short chase times meant that
only very small DNA fragments were synthesized along the lagging strand. As
the chase got longer and the DNA had more time to replicate, the pieces of
the lagging strand began to integrate into longer, heavier and faster
sedimenting DNA strands. Scientists now know that Okazaki fragments in
bacterial DNA are typically 1,000 to 2,000 nucleotides long, whereas in
eukaryotic cells they are only about 100 to 200 nucleotides long.
Termination
DNA replication termination occurs when two replication forks meet on the
same stretch of DNA, and the following events occur, but not necessarily in
that order: The forks converge until all the DNA in between has been
unwound. Fill the remaining gap and ligate. Catenanes are removed.
Replication proteins are unloaded.
Summary
The study of DNA replication began shortly after the structure of DNA was
elucidated and continues to this day. Although the stages of initiation,
unwinding, primer synthesis, and elongation are now understood in their
simplest terms, many questions remain unsolved, especially when it comes to
replication of eukaryotic genomes. Scientists have been dedicated to the
study of replication for decades, and researchers such as Kornberg and
Okazaki have made many important breakthroughs. Yet there is still much to
learn about replication, including how errors in this process contribute to
human disease.