DNA Replication

The entire DNA of a cell is replicated during the S (synthesis) phase of the cell cycle. The principle of replication is based on complementary nucleotide base pairing: adenine forms hydrogen bonds with thymine (or uracil in RNA) and guanine forms hydrogen bonds with cytosine. Replication occurs prior to cell division in the S phase of the cell cycle in order to allow 2 sets of chromosomes to be present during the metaphase of mitosis, after which they are equally partitioned into 2 new cells during separation (anaphase).

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Preparation and Prerequisites for Replication

  • Replication occurs during the S (synthesis) phase of the cell cycle.
  • The duration of these cell cycle phases varies considerably in different kinds of cells. For a typical rapidly proliferating human cell with a total cycle time of 24 hours, the G1 (growth) phase might last about 11 hours, S phase about 8 hours, G2 (growth and preparation for mitosis) about 4 hours, and M (mitosis) about 1 hour.
  • The DNA of a human cell mostly exists as 46 chromosomes (23 pairs = 2n) within the nucleus. A small fraction of the total is present as mitochondrial DNA, which has approximately 16,500 base pairs containing 37 genes, all of which are essential for normal mitochondrial function.
Simple cell cycle dna

Cell cycle, consisting of G1, S, G2, and M phases

Image: “The four phases of the cell cycle“ by Simon Caulton. License: CC BY-SA 3.0

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Initiation

  • Origins of replication: Certain proteins recognize sections of DNA (AT-rich) from which replication can begin.
    • Prokaryotes possess only a single origin of replication.
    • Eukaryotes have multiple origins of replication. In human DNA, there are more than 30,000 origins of replication, without which the S phase would last about 40 times longer.
  • In prokaryotes, a protein called DnaA binds to an origin of replication. In eukaryotes, this is performed by the origin recognition complex (ORC), a 6-unit DNA binding complex.
  • ATP-dependent helicase DnaB (prokaryotes): unravels the DNA double helix, which separates the 2 strands to expose 2 single strands.
    • Helicase (in eukaryotes): moves in the 5’ → 3’ direction along the DNA molecule and forms the replication fork by using energy from ATP hydrolysis to break the hydrogen bonds between annealed nucleotide bases of the complementary strands, thereby separating them
    • Bloom’s syndrome is a cancer predisposition disorder caused by mutations in the BLM helicase gene, 1 of the nearly 100 helicases present in humans.
  • Single-strand binding proteins (SSB): bind to the unraveled single strands and prevent them from re-attaching to each other after they have been separated by helicase
  • As the 2 strands of template DNA unwind (unravel), the DNA ahead of the replication fork is forced to rotate in the opposite direction so that the DNA becomes twisted around itself without the presence of topoisomerases, which catalyze the reversible breakage and joining of DNA strands. The transient breaks produced by these enzymes allow the 2 strands of DNA to rotate freely around each other so that replication can proceed without causing undue torsional stress on the DNA template, which could lead to breakage of the nucleic acid strands.
    • In prokaryotes, fluoroquinolone antibiotics inhibit topoisomerases II (DNA gyrase) and IV.
    • In eukaryotes, the chemotherapeutic agents irinotecan and topotecan inhibit topoisomerase I, whereas etoposide and teniposide inhibit topoisomerase II.
DNA Replication

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.

Image: “DNA Replication” by Mariana Ruiz. License: Public Domain
PolymeraseFunctionExonuclease activity
αSynthesizes the RNA primer and initiates DNA synthesis along the lagging strand3’ → 5’
βRepairs DNANone
γReplicates mitochondrial DNA3’ → 5’
δSynthesizes the lagging strand, filling DNA gaps after removal of primer3’ → 5’
εSynthesizes the leading strand3’ → 5’ and 5’ → 3’

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Elongation

  • A primer is necessary for the synthesis of daughter strands of DNA
    • It is a 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 the leading strand and discontinuously on the lagging strand.
  • Leading strand
    • Replication continuous due to free 3’ OH group
    • Requires only 1 primer
  • Lagging strand
    • Replication discontinuous to ensure always a free 3’ OH group for elongation
    • Forms Okazaki fragments (1,000–2,000 nucleotides in length)
    • A primer is needed for each segment of DNA.
  • Proceed in 5’ → 3’ direction
  • Base pairs are added to the free 3’ OH end of the daughter strand.
  • Catalyzed by DNA polymerase (polymerase δ in eukaryotes and DNA polymerase III in prokaryotes)
    • Forms 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 the 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
    • Gaps filled with complementary deoxynucleotides
    • Prokaryotes: DNA polymerase I adds deoxynucleotides 1 at a time then proofreads.
    • Eukaryotes: DNA polymerase δ
  • Joining the ends
    • DNA ligase joins the free ends of the daughter strands.
    • 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)

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 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 fragments at 3′ ends of chromosomes
    • Each telomere consists of several thousand base pairs (tandem repeats of TTAGGG)
    • Function of a telomere:
      • Prevents the loss of structural genes
      • The lagging strand becomes shorter after each round of replication due to the removal of the RNA prime so DNA is lost from the telomere rather than from the coding sequence.
      • Functions like a cellular clock since the cell undergoes replicative senescence or apoptotic cell death when the telomere length goes below a critical limit. The length of telomeric DNA determines the lifespan of a cell in culture.
  • Telomerase
    • Special reverse transcriptase that carries its own RNA template
    • Maintains telomeres
    • Present in rapidly dividing cells, embryonic and cancer cells
      • Activity is especially enhanced in cancer cells so their telomeres stay long and they can keep dividing without regard to a molecular clock even if they reach the end of their normal lifespan.

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