ZOOHCC - 501: Molecular Biology (Theory)
Unit 2: DNA Replication
RNA Primer:
A primer is a short nucleic acid sequence that provides a starting point
for DNA synthesis. In organisms, primers are short strands of RNA. Primers
must be synthesized by an enzyme called primase, a type of RNA polymerase,
before DNA replication can occur. Synthesis of primers is necessary because
DNA syntheses, called DNA polymerases, can only add new DNA nucleotides to
an existing strand of nucleotides. Thus, primers help prepare and lay the
groundwork for DNA synthesis. Before DNA replication is complete, primers
are removed and sequence gaps are filled with DNA by DNA polymerase. In the
laboratory, scientists can design and synthesize DNA primers with specific
sequences that bind to sequences on single-stranded DNA molecules. These DNA
primers are commonly used to perform the polymerase chain reaction for
copying DNA fragments and DNA sequencing.
RNA Primer:
An RNA primer is a short sequence of RNA that serves as a starting point
for DNA synthesis. It is synthesized by an enzyme called primase, which is
a type of RNA polymerase.
During DNA replication, the double-stranded DNA is unwound by the
helicase enzyme. The leading strand is synthesized continuously by DNA
polymerase, while the lagging strand is synthesized in short fragments
known as Okazaki fragments. In both cases, the DNA polymerase requires a
3'-OH group to add new nucleotides to the growing strand.
However, DNA polymerase cannot initiate synthesis on a bare DNA template;
it requires a pre-existing 3'-OH group to start adding nucleotides. This
is where the RNA primer comes in. Primase adds a short RNA sequence (about
10 nucleotides long) complementary to the template DNA strand, which
provides a 3'-OH group for DNA polymerase to start adding
nucleotides.
Once the RNA primer is synthesized, DNA polymerase takes over and begins
to extend the primer by adding DNA nucleotides to the 3'-OH group. Later,
the RNA primer is removed by the exonuclease activity of DNA polymerase,
and the resulting gap is filled by DNA synthesis.
Okazaki fragments
Okazaki fragments are short DNA stretches formed during discontinuous
synthesis of the lagging strand during DNA replication. It is essential as
it allows the synthesis of her two daughter strands required for cell
division.
Discrete replication produces Okazaki fragments The Okazaki fragment in
bacteria and bacteriophage T4 is 1000-2000 nucleotides long, whereas in
eukaryotes it is only about 100-300 nucleotides. Each Okazaki fragment is
primed with a short RNA because DNA polymerase cannot initiate DNA
synthesis. Coordination of leading and lagging strand replication and
synthesis of RNA primers for lagging strand replication are discussed
elsewhere in this encyclopedia. In some organisms, such as Escherichia
coli and bacteriophage T4, the same DNA polymerase is responsible for both
leading and lagging strand DNA replication. Yeast, and probably all
eukaryotes, have different DNA polymerases that lead and retard the
replication of strand DNA. The DNA polymerase epsilon (ε) is primarily
responsible for leading strand replication, while the DNA polymerase delta
(δ) is responsible for Okazaki fragment synthesis and lagging strand
replication. Joining of the Okazaki fragment requires removal of the RNA
primer, DNA replication to complete the synthesis, and treatment of the
ends by a nuclease to create a 'nick' that can be closed by the action of
DNA ligase.
Explanation:
Primer RNA is the RNA that initiates DNA synthesis. DNA synthesis requires
a primer because no known DNA polymerase can initiate polynucleotide
synthesis. DNA polymerases specialize in extending polynucleotide chains
from available 3' hydroxyl ends. In contrast, RNA polymerase can extend
and initiate polynucleotides. Primase is a specialized RNA polymerase that
synthesizes short-lived oligonucleotides that are used only during DNA
replication. "Transcriptional" RNA polymerases primarily synthesize
messenger RNA, but transcripts are sometimes used to initiate DNA
synthesis. For example, the single-stranded DNA phage M13 genome uses RNA
polymerase instead of primase to initiate DNA synthesis. Furthermore, a
leading hypothesis for the initiation of mitochondrial DNA replication is
that mitochondrial RNA polymerase synthesizes polymers that do not migrate
from the template. A specialized RNase MRP then cleaves the ribopolymer at
specific positions, leaving the exposed 3'-hydroxyl terminus to act as a
primer for DNA synthesis. Finally, transfer RNAs constitute a special
class of primer RNAs, as specific tRNA species are used by retroviral
reverse transcriptase to initiate replication of the retroviral genome. It
is also possible to initiate DNA synthesis without primer RNA. Adenovirus
and the ϕ29 initiator protein covalently bind to the 5' ends of both
linear duplex DNAs, providing serine β-hydroxy groups for extension by DNA
polymerases. Another example is that many plasmids encode
sequence-specific nucleases that cleave one strand of the duplex to
generate the 3' hydroxyl for host DNA polymerases. An example of an animal
virus is a parvovirus in which the 3' end of the parental strand forms a
DNA hairpin and primes its complement.
Discovery of the Okazaki Fragment:
Discovery of the Okazaki Fragment
The Okazaki fragment was detected by pulse labeling of E. coli with 3
H-thymidine under conditions that significantly reduced the rate of
cell growth and division. To uniformly label DNA with 14C, different
generations of E. coli were cultivated at the optimum temperature of
37°C
in the presence of 14C-thymidine. Cells were then cooled to 20° C. and
pulsed with 3 H-thymidine for 10 seconds to label nascent DNA under
conditions in which a decrease in DNA replication rate reveals the
presence of transient intermediates.
The doubling time of E. coli is about 40 minutes at 37°C and about 250
minutes at 20°C. In pulse-chase experiments, cells were supplemented
with large amounts of unlabeled thymidine and pulse-labeled for 10 s at a
temperature of 20 °C. Incubation continued for the suggested time. Neat
cellular DNA was then isolated, fractionated by sedimentation on an
alkaline sucrose gradient, and completely denatured.
The amount of acid-insoluble radioactivity was quantified in each
section of the gradient that could be dissolved by treatment with
deoxyribonuclease. Under these conditions, the majority of 3H-DNA
initially appears as fragments of approximately 50–5000 nucleotides in
length, followed by rapidly elongated 3H-DNA fragments, some of which
are involved in DNA replication. Ordered as a temporary intermediate.
