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.