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DNA Replication Part II

Notes

DNA Replication Part II

Sections

DNA Replication

  • Complementary
  • Semiconservative
  • Bidirectional
  • Occurs in the S phase
  • Synthesis in the 5' to 3' direction – antiparallel

KEY ENZYMES

  • Helicase separates DNA strands at replication fork
  • Topoisomerase relieves supercoiling downstream of replication fork
  • Primase synthesizes RNA primer
  • DNA polymerase synthesizes DNA
  • Ligase joins DNA fragments together

Key differences between Prokaryotes and Eukaryotes

• Eukaryotes have multiple origins of replication
• Different DNA polymerases, helicases, topoisomerase and ligase
• Eukaryotes have nucleosomes and telomeres
• Eukaryotes typically have more DNA

Prokaryotic DNA Polymerases

• DNA pol III: synthesizes both the leading and lagging strands.
• DNA pol I: removes RNA primers and fills the remaining gaps with DNA nucleotides.

Eukaryotic DNA polymerases

See end of tutorial for scientific update regarding DNA pol epsilon and DNA pol delta.

• DNA pol epsilon: elongates leading strand DNA.
• DNA pol delta: elongates lagging strand DNA and displaces RNA primers from the lagging strand.
• DNA pol alpha: complexes with primase to synthesize primers that comprise RNA and DNA.

Nucleosomes

  • Comprised of histones and DNA: package DNA compactly
  • Disassemble when replication fork approaches and reassembled in its wake

Telomerases

  • Add guanine-rich telomeric repeats to the 3' end of parent strand
  • Telomeric repeats form loop that protects the DNA from degradation or damage

CLINICAL CORRELATION

Telomere length

  • Associated with aging
  • After birth: active telomerase limited to germ and stem cells in humans
  • Telomeres shorten with each DNA replication = finite number of DNA replications

Full-Length Text

  • Here we will learn about DNA replication in eukaryotes and highlight key differences between the eukaryotic and prokaryotic processes.
    • This is part two of a two part tutorial. In part one, we learned DNA replication in prokaryotes, using E. Coli as a model.

We've removed our topoisomerase diagram to give us some extra room.

First, let's add the key differences between eukaryotic and prokaryotic replication.

  • Denote them as follows:
    • Origins of replication –eukaryotes have multiple, whereas prokaryotes, such as E. coli, only have one.
    • Polymerases – eukaryotes and prokaryotes use different polymerases.
    • Nucleosomes – eukaryotes have them, prokaryotes do not.
    • Telomeres – eukaryotes have them, whereas prokaryotes do not.

Now, let's illustrate eukaryotic DNA replication and address each of these key differences.

  • To begin, draw a double-stranded DNA molecule with multiple origins of replication, eukaryotes have much more DNA than prokaryotes, and thus multiple origins.
  • Use arrows to illustrate that DNA replication is bidirectional at each origin.

Now, let's transition to a detailed view of the replication fork.

  • To begin, recall the key molecules involved in both prokaryotic and eukaryotic replication:
    • Helicase is an enzyme that catalyzes DNA strand separation.
    • Topoisomerase alleviates the super-coiling of DNA downstream of the origin.
    • Primase synthesizes short fragments of RNA.
    • DNA polymerase synthesizes DNA. There are multiple DNA polymerases, each with a specialized function.
    • Ligase ligates fragments of DNA together during replication.

Let's use the previously drawn replication fork to highlight key molecular differences between prokaryotic and eukaryotic DNA replication.

  • First, indicate prokaryote and eukaryote in different colors.
  • Label the following prokaryotic homologs in our diagram:
    • DNA polymerase III, which synthesizes both the leading and lagging strands.
    • DNA polymerase I, which removes RNA primers and fills the remaining gaps with DNA nucleotides.
  • Label the following eukaryotic homologs:

See end of tutorial for scientific update regarding DNA pol epsilon and DNA pol delta.

  • DNA polymerase epsilon, which elongates leading strand DNA.
  • DNA polymerase delta, which elongates lagging strand DNA and displaces RNA primers from the lagging strand.
  • DNA polymerase alpha, which complexes with primase to synthesize primers that comprise RNA and DNA.

To emphasize this point, recall that in prokaryotes primase synthesizes RNA-only primers.

  • Note that prokaryotes and eukaryotes also differ in their helicases, topoisomerases and ligases.
    • We focus on polymerases because they play functionally distinct roles in prokaryotes and eukaryotes.
  • As a clinical correlation, write that chemotherapeutic drugs often inhibit DNA replication in cancer cells.
    • For example, nucleoside analogs are drugs that incorporate into daughter strands and terminate their elongation.

Now, let's address eukaryotic nucleosomes, which comprise histones.

  • Draw a replication fork, and this time include the double helix structure.
  • Draw nucleosomes downstream of the advancing replication fork; these histone proteins must be removed before replication can occur.
  • Draw nucleosomes upstream of the replication fork.
  • Show that they associate with both the parent strands and the newly synthesized daughter strands.
    • Histones reassemble behind the replication fork.
  • Indicate that Okazaki fragments approximate the length of DNA wrapped around nucleosomes.
  • There are a number of eukaryote-specific proteins associated with the displacement and reformation of nucleosomes during DNA replication. We will not discuss them here.

Finally, let's illustrate telomeres, the last key difference between eukaryotic and prokaryotic replication.

  • Draw a strand of DNA and label it the lagging strand.
  • Draw an incomplete daughter strand below it.
  • Label the 5-prime and 3-prime ends of the parent strand and then the daughter strand.
  • Show that an enzyme called telomerase adds short guanine-rich repeats (called telomeric repeats) to the three-prime end of the parent strand.
    • Telomerase is active in all cells before birth, but inactive in most somatic cells after birth.
  • Show that the telomeric repeats create space for a primer to bind.
    • The number of repeats ranges from two or three in the simplest eukaryotes to almost thirty in humans.
  • Illustrate that DNA polymerase delta synthesizes the remaining portion of the daughter strand.
    • Thus, telomeres prevent the loss of genetic material during replication.
  • Show that after DNA polymerase delta displaces the primer and ligase fills the gap, the telomeric repeats form a loop at the end of the chromosome.
    • This protects the end of the chromosome from degradation or damage.
    • We discuss t-loop formation elsewhere.
  • As a clinical correlation, denote that telomere length is associated with the aging process.
    • After birth, active telomerase is largely limited to germ and stem cells.
    • Thus, telomeres shorten with each DNA replication resulting in a finite number of DNA replications.

Scientific Update

  • Since the creation of this tutorial, it is not accepted that DNA pol delta carries out both leading and lagging strand DNA synthesis, rather than Pol delta replicating the lagging strand and pol epsilon replicating the leading strand.
    • Johnson, Robert E., Roland Klassen, Louise Prakash, and Satya Prakash. "A Major Role of DNA Polymerase δ in Replication of Both the Leading and Lagging DNA Strands." Molecular Cell 59, no. 2 (July 16, 2015): 163–75. https://doi.org/10.1016/j.molcel.2015.05.038.

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