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DNA Replication Before a cell divides--either sexually or asexually--it must duplicate its genetic material.
Many investigators contributed to our understanding of DNA replication

DNA Replication is Semiconservative
In 1958, Matthew Meselson and Francis Stahl published their work on DNA replication.

They used density gradient centrifugation, an in vitro technique they invented

  • Label DNA molecules with (heavy) 15N
  • Hybridize DNA strands
  • Allow them to replicate
  • Centrifuge molecules, separating them by density

They found that DNA replication is semi-conservative:
each new DNA strand consists of

  • half the original template
  • half new material

Visualizing DNA Replication
In 1963, J. Cairns published his autoradiographs of semiconservative replication in E. coli.

During circular DNA replication

  • the double helix is nicked at the origin site
  • The strands are replicated as they separate, forming a "theta"
  • Cairns named the mid-replication E. coli DNA theta structure.
  • This type of replication is known as displacement replication

DNA replication is bidirectional.
It proceeds in both directions around the circle, forming a single replicon

A replicon is a region where DNA is separated and synthesis is taking place.

Small genomes such as those of

  • plasmids
  • mitochondria
  • chloroplasts
can replicate either via displacement replication or rolling circle replication

In the rolling circle replication

  • one strand is replicated continuously
  • the other strand is replicated discontinuously
  • This allows the quick manufacture of many copies of the genome.

    In eukaryotes, the replicating chromosome consists of multiple replicons along its length.

DNA Replication
The DNA replication process described here was discovered in E. coli.
The process is very similar, though somewhat more complicated, in eukaryotic cells.

The Replisome
The DNA/enzyme conglomerate represented in these images and animations is known as a replisome.

The replisome is a biological machine responsible for replicating DNA.
Its components:

  • Helicase breaks hydrogen bonds between the complementary DNA strands and unwinds the double helix.
  • Primase lays down an RNA primer on the DNA template.
    • Helicase and primase bind together to form the primosome during DNA replication.

  • Gyrase nicks the DNA strands just ahead of the replication fork, relaxing positive supercoiling

  • DNA polymerase III synthesizes new DNA along the leading and lagging strands.
    • DNA polymerase III is a large, ring-shaped enzyme.
    • It forms a dimer, each half of which encircles one of the two template strands.
    • One half of the dimer synthesizes new DNA on the leading strand
    • The other half of the dimer synthesizes new DNA on the lagging strand

  • DNA polymerase I removes RNA primers and replaces them with DNA nucleotides, and serves in proofreading and repair.

  • DNA polymerase II is responsible for proofreading and repair
    • DNA polymerase active sites are configured to add nucleotides only in the 5' to 3' direction.
    • The 5' end of the new, incoming nucleotide is attached to the 3' end of the previously attached nucleotide on the replicating strand.

  • Ligase catalyzes phosphodiester bonds between unbonded 3' and 5' DNA fragments (e.g., Okazaki fragments)

Animation: DNA Replication Overview
Animation: DNA Replication Close up

DNA Replication, Step by Step
The processes described in the following section are seen in prokaryotic cells.
The basic chain of events is very similar in eukaryotes.
We will enumerate differences between prokaryotic and eukaryotic DNA replication at the end.

Initiation of DNA Replication
In E. coli, origin of replication is a ~245bp gene named oriC. 1. OriC is recognized and bound by initiator proteins which
  • recognize oriC
  • recruit replication factors
  • begin to denature the DNA double helix
  • promote attachment of the primosome made of
    • helicase (unwinds the double helix and moves the primosome along the DNA)
    • primase (lays down RNA primer)

2. Helix destabilizing proteins (a.k.a. single-strand binding proteinsattach to the denatured strands, preventing them from re-annealing.

3. The Y-shaped region where DNA is separated at each ends of the replicon is called a

  • replication fork or
  • Y junction

Elongation of New DNA Strands
DNA polymerase III binds to the template strand and begins laying down complementary DNA nucleotides.

Nucleotides can be attached only in the 5' to 3' direction.

  • The 5' 3-phosphate end of the incoming nucleoide is oriented towards the 3' end of the nucleotide to which it will be attached.
  • Once attached, the 3' hydroxyl end of the newly added nucleotide is available for binding to the 5' end of the next incoming nucleotide.

Because synthesis is bidirectional, a complication arises at the Y junctions.

The two separated template DNA strands are named the

  • leading strand (shown at the left)
    • The 3' end of the already-attaced nucleotide points towards the growing replication fork.
    • The 5' end of the incoming nucleotide can be attached via continuous replication.

  • lagging strand
    • The 3' end of the already-attaced nucleotide points away from the growing replication fork.
    • This presents a complication, requiring discontinuous replication.

      Continuous and discontinuous replication occur simultaneously on opposite template strands.

Discontinuous Replication
On the lagging strand, DNA must be replicated in small segments.

Primase manufactures an RNA primer just downstream of the Y junction.

  • The 3'-OH end of the primer points away from the Y junction.
  • The 5' end of the primer points towards the Y junction.
    • Thus, nucleotides can be added to elongate the strand only away from the junction.

    • As Y junction widens, primase lays down another RNA primer.

    • When DNA polymerase III meets the primer of the previous Okazaki fragment, it detaches, ending synthesis of that fragment.

    • It then attaches to the next primer to make another Okazaki fragment.

    • The result: adjacent Okazaki fragments are laid down as DNA polymerase manufactures them.

  • Termination of DNA Replication: Ligation
    On a circular genome, DNA polymerase III will eventually meet the 3' end of the first nucleotide.

    It will detach after adding the final nucleotide, its 5' end next to the 3' of the first nucleotide.

    DNA polymerases cannot catalyze the reaction necessary to join these last two nucleotides.

    This job is done by DNA ligase.

    The final phosphodiester bond is made via ligation.

    Ligase also joins adjacent Okazaki fragments, completing the lagging strand.,

    DNA Polymerases: Form and Function

    DNA polymerase III is a large, complex enzyme consisting of 17 polypeptide subunits.
  • The polymerization core consists of the subunits
    • α - the polymerase
    • ε - the 3'--> 5' exonuclease
    • θ - stimulates the ε subunit

  • The θ subunit causes the two parts of the DNA polymerase to dimerize (i.e., form a two-part holoenzyme).

  • Two β subunits form a "sliding clamp" that hold the enzyme onto the DNA strand and keep it from disengaging. This is critical to the processivity of the molecule.

  • The remaining subunits apparently serve a clamp loader function, helping to attach the β sliding clamps to the DNA polymer.

    DNA polymerases can not only add nucleotides in the 5' to 3' direction.
    They also can remove them in the 3' to 5' direction.

    • exonucleases remove nucleotides from the end of a nucleic acid chain.

    • endonucleases remove nucleotides from the middle of a nucleic acid chain by breaking the sugar/phosphate backbone on either side of the nucleotide(s) to be removed.

    Exo- and endonuclease action are also important to proofreading and repair of the new chain.
    If a DNA polymerase detects an error, it can snip it out and correct it.

  • Topoisomers of DNA
    When not replicating, DNA exists in a "relaxed" form.

    During replication, however, DNA can become supercoiled:

    • Positive supercoiling occurs when the chromosome twists in the same direction as the DNA helix, iuncreasing overall coiling)

  • Negative supercoiling occurs when the chromosome twists in the opposite direction of the DNA helix, relaxing coiling.

    Positive supercoiling causes problems, as demonstrated in the video to the left.

    Enzymes that uncoil topoisomers of DNA are known as topoisomerases. Some of these attach and operate just in front of the Y-junction during DNA replication.

    • Type I topoisomerases snip one DNA strand and insert it through the other strand's loop to relax the coil.

    • Type I topoisomerases snip both DNA strands and pass them through the resulting gap to relax the coil.

    DNA gyrase is a type II topoisomerase unique to bacteria.
    Because is it not found in eukaryotes, it can be used as a target for anti-bacterial agents.

    For example, the fluoroquinolone class of antibiotics

  • enrofloxacin
  • ciprofloxacin
  • marbofloxacin target and inactivate DNA gyrases.

  • Bacteriocidal agents kill bacteria outright.
  • Bacteriostatic agents slow bacterial growth.
  • Either type will help an organism fight a bacterial infection until its own immune system can take over and finish the job.

  • DNA Replication in Eukaryotes
    DNA replication in eukaryotes is essentially similar to that seen in prokaryotes, though the biological machinery is more complex. For example, there are at least 15 DNA polymerases, each with specific functions.

    In eukaryotic DNA replication...

    The Trouble With Telomeres
    Telomeres are long, repeating base sequences at the ends of each chromosome.
    They are crucial to the life and longevity of the cell.
    • "Capped" and unreactive, they keep the ends of the chromosomes in the cell from accidentally reacting with each other and becoming stuck to each other.

    • The human telomere consists of as many as 2000 repeats of the sequence 5' TTAGGG 3'.

    • Understand the telomere and see the possible link to cancer therapies of the future.

    • DNA Polymerases cannot create telomeres.

    • If nothing is added to the terminal end of newly replicated DNA, then chromosomes become successively shorter with each replication.

    Carol Greider and Elizabeth Blackburn showed that telomerase, an enzyme composed of protein and RNA, can maintain chromsome length by adding telomeres to the ends of newly replicated eukaryotic chromosomes.

  • The RNA portion of telomerase contains a base sequence complementary to the terminal DNA repeat

  • Telomerase binds to the hanging bit of DNA and adds bases complementary to its own RNA strand.

  • The remaining gap can then be filled in by DNA polymerase.

    In humans (and probably other vertebrates and mammals), telomerase is active only in the germline.
    It is not present in most somatic cells.
    But it is active in the germline.

    Investigators were surprised to find that active telomerase is found in many types of tumor cells. A cancerous cell evidently produces telomerase after it becomes cancerous.

    • Cancer cells often already have very short telomeres.
    • The abnormal re-production of telomerase allows these faulty cells to maintain their telomeres.
    • This can contribute to the cell's immortality.

  • Eukaryotic Finale: Assembling the Nucleosomes
  • Histones are manufactured while DNA is being replicated, during S phase of interphase.
  • Histone synthesis ceases shortly before DNA replication is complete.
  • DNA is wound around histones as as it is replicated, though there are short regions of histone-free DNA just around the replication fork.
  • Nucleosome packaging of DNA is be involved in epigenetic inheritance.