DNA replication is the process by which DNA creates a copy of itself for cell division. Replication occurs prior to cell division in the S phase of the cell cycle. This creates two sets of chromosomes for the anaphase of mitosis, during which the duplicated genetic material is equally distributed to opposite poles of the cell. The following telophase leads to the formation of two new cells. The principle of replication can be extrapolated from complementary base pairing (adenine binds with thymine/uracil, guanine binds with cytosine).

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DNA replication

Image: “ DNA replication or DNA synthesis is the process of copying a double-stranded DNA molecule. This process is paramount to all life as we know it. License: Public Domain


Preparation and Prerequisites for Replication

  • Replication occurs during the S phase of the cell cycle.
  • In humans, this phase takes about 8–12 hours.
  • DNA of a human cell exists as 46 chromosomes (2n).

Video Gallery

DNA Replication by Kevin Ahern, PhD

Cell Cycle simple

Image: Cell cycle, consisting of G1, S, G2, and M phases by Simon Caulton. License: CC BY-SA 3.0

Initiation

  • Origins of replication (ori): Certain proteins recognize sections of DNA (AT-rich) from which replication can begin.
    • Prokaryotes: only a single ori
    • Eukaryotes: multiple oris
      • Human DNA: contains over 30,000 oris without which the S phase would last about 40 times longer
  • In prokaryotes: the protein DnaA binds to an ori (in eukaryotes, this is done by the origin recognition complex, ORC)
  • ATP-dependent helicase DnaB (prokaryotes): unravels the DNA double helix, which separates the two strands to expose two single strands.
    • Helicase (eukaryotes): moves in the 5’-3’ direction along the DNA molecule and forms the replication fork by forcing the complementary strand apart.
      • Deficient in Bloom syndrome (BLM gene mutation)
  • Single-strand binding proteins (SSB): bind to the unraveled single strands and prevent them from re-attaching onto each other behind the helicase molecule
  • Section before the replication fork that is unwound during replication rotates during the process.
  • Topoisomerases: prevent torsional stresses and as a result protects the DNA from unwanted breaks by unraveling supercoiled DNA.
    • In prokaryotes, fluoroquinolones inhibit topoisomerases II (DNA gyrase) and IV.
    • In eukaryotes, irinotecan/topotecan inhibits topoisomerase I; whereas etoposide/teniposide inhibit topoisomerase II

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Replication Fork by Kevin Ahern, PhD

DNA replication

Image: DNA replication by LadyofHats. License: Public Domain

Table: DNA elongation in eukaryotic cells involving a variety of proteins and enzymes

Polymerase Function Exonuclease activity
α Synthesizes the RNA primer, initiations DNA synthesis, and the lagging strand 3’ to 5’
β Repair DNA None
γ Replicate mitochondrial DNA 3’ to 5’
δ Synthesizes the lagging strand, filling DNA gaps after removal of primer 3’ to 5’
ε Synthesizes leading strand 3’ to 5’ and 5’ to 3’

Elongation

  • Primers: necessary for synthesis of daughter strands of DNA
    • Short piece of RNA (8–10 base pairs)
    • Complementary to template strand
    • Synthesized by primase (a DNA-dependent RNA polymerase)
      • In eukaryotes, it is a subunit of DNA polymerase alpha
  • DNA synthesis
    • Replication occurs continuously on leading strand and discontinuously on lagging strand
  • Leading strand
    • Replication continuous due to free 3’ OH group
    • Requires only one primer
  • Lagging strand
    • Replication discontinuous to ensure always a free 3’ OH group for elongation
    • Form Okazaki fragments (1000-2000 nucleotides in length)
    • Primer for each segment of DNA
  • Proceed in 5’ → 3’ direction
  • Base pairs added to free 3’ OH end of daughter strand
  • Catalyzed by DNA polymerase (polymerase δ in eukaryotes and DNA polymerase III in prokaryotes)
    • Form ester bond between 3’OH group to α-phosphate of nucleotide
    • Pyrophosphate released
  • Proofreading
    • Accomplished only by polymerases with 3’ → 5’ exonuclease activity
  • Primer removal
    • Excised in opposite direction of synthesis (i.e., 5’ → 3’)
    • Prokaryotes: by RNase H and the 5’ → 3’ exonuclease activity of DNA polymerase I
    • Eukaryotes: by FEN-1 (flap endonuclease-1)
  • Filling the gaps
    • Fill gaps with complementary deoxynucleotides
    • Prokaryotes: DNA polymerase I adds deoxynucleotides one at a time then proofreads
    • Eukaryotes: DNA polymerase δ
  • Joining the ends
    • DNA ligase joins free ends of daughter strand
    • Reaction involves:
      • Transfer of AMP to 5’ phosphate end
      • AMP is cleaved and 5’ phosphate end bound to 3’ OH end of other fragment
  • After the duplication of the DNA, there are 46 double chromatid chromosomes (4n)
    • Following anaphase and cytokinesis (of mitosis) reduced to 46 single chromatid chromosomes (2n)

Video Gallery

DNA Polymerase by Kevin Ahern, PhD
Other Replication Proteins by Kevin Ahern, PhD
Leading and Lagging Strand by Kevin Ahern, PhD

Termination

  • Initiated by binding of termination proteins (ter proteins) to termination sequences.
  • Different termination in prokaryotes (circular DNA) and eukaryotes (linear DNA)
  • Eukaryotic chromosomes → linear
    • A gap will exist
  • If during replication the complementary primer is at the 5’-OH end of the daughter strand, there is no free 3’-OH end for ligation.
  • The gap which is left by the primer cannot be filled—this means that after each replication, a small piece at the end of the DNA is missing.
  • This is the reason for a non-coding repetitive sequence (GGGTTA) with over 10,000 base pairs at the end of the eukaryotic chromosome (telomere).
  • Coding sequences, genes, only stop being completely replicated after 30-50 cell cycles; this limits the life expectancy of most somatic cells.

Telomeres and Clinical Relevance

  • Telomeres
    • Non-coding DNA fragment
    • Consist of several thousand base pairs (tandem repeats of TTAGGG)
    • At 3’ end of chromosome
    • Function:
      • Prevent loss of structural genes in linear DNA
      • Lagging strand become shorter each round of replication due to the removal of RNA primer→ lose telomere rather than coding sequence
      • Function as cellular clock
  • Telomerase
    • Special reverse transcriptase that carries own RNA template
    • Maintains telomeres
    • Present in rapidly dividing cells, embryonic and cancer cells

Video Gallery

Telomeres by Kevin Ahern, PhD

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