DNA Replication
VISUALIZE FIRST!!
https://www.youtube.com/watch?v=9kp9wiYMQUU
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Before we get into the process of replication….
“Unzipped DNA”
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- Recall the structure of DNA
- DNA is in an ”anti-parallel”
orientation (3’ to 5’ alignment)
- Chargaff’s Rule: Adenine is
proportional to Thymine, and
Guanine is proportional to Cytosine.
- Watson & Crick: Proved that
Thymine and Adenine Hydrogenbond with each other while Guanine
and Cytosine Hydrogen bond (making
up the “rungs” of the DNA ladder)
- Therefore A-T and G-C combine.
- 3 H-bonds between G-C
- 2 H-bonds between A-T.
When, Where & Why Does DNA Replication Occur?
- WHEN? Interphase is divided into
3 stages
- G1: Cell is growing
- S Phase: S stands for “synthesis”.
DNA replication takes place here in
Interphase, prior to mitosis or
meiosis.
- WHERE? In the nucleus
- WHY? To produce an identical
copy of DNA (using the original as a
template) so each daughter cell
possesses the same genetic
information as the parent cell.
Preparing for DNA Replication
Step 1:
- The DNA molecule must unzip.
- The H-Bonds between the base pairs must be broken
- DNA Helicase is responsible for this process
- Active site of DNA Helicase is specific for a region of DNA called the “replication
origin”
- Different organisms (eukaryotes and prokaryotes) have different combinations of
nucleotides that form the active site for DNA helicase.
- Bacteria have one big circular loop of DNA (not a complex double helix with
multiple chromosomes…) – Therefore ONE DNA Helicase can bind to ONE
replication origin and unzip the entire molecule (does not take too long).
- However, Eukaryotic cells need MULTIPLE replication origins in DNA to speed up
the unzipping process with MULTIPLE DNA Helicase enzymes working!
Helicase:
- Moves in ONE direction only, away from the
replication fork, starting at the replication
origin.
- The Active site recognizes as specific sequence
of nucleotides.
- As Helicase moves along, it breaks H-bonds
between base pairs.
- There is ONE replication origin in prokaryotes.
- There are MANY replication origins in
eukaryotes.
Direction
The Unzipping Process
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Step 2: Single Strand Binding Proteins (ssbp’s)
- What prevents H-bonds from RE-forming between
base pairs after DNA Helicase moves down the
line?
- The process of re-forming H-bonds in DNA is called
Reannealing.
- To prevent reannealing, ssbp’s temporarily bind to
the nitrogen bases to prevent the reforming of Hbonds.
- This keeps the two strands separated long enough
for DNA replication to occur.
Direction
Preparing for DNA Replication
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Step 3: DNA Gyrase
- DNA Gyrase binds to the DNA
molecule upstream from DNA
Helicase.
- Temporarily breaks phosphodiester
bonds between nucleotides.
- This “cuts the rails” of the DNA
molecule to release tension caused by
unzipping, and then RE-forms these
bonds in the exact same locations.
Direction
Preparing for DNA Replication
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DNA Replication
DNA Polymerase III:
- Main working enzyme of DNA replication. The reason
it is labeled “III” is because it was discovered 3rd
- Good enzyme name: Creates a polymer of nucleotide
- Requirements to BIND and work:
- Needs a pre-existing strand of DNA to act as a
template
- Needs to have nucleoside building blocks with 3
phosphates (dATP, dTTP, dCTP, dGTP). “d” =
“deoxy”. ATP = Adenosine Triphosphate… etc.
- A short RNA primer strand with a free 3’ OH end.
(10-60 nucleosides long, synthesized by PRIMASE)
NOPE!!!
NOPE!!
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DNA Polymerase III: A closer look
- DNA Polymerase III has an active site for a 3’
hydroxyl and for the the DNA template strand.
- Shown here: DNA Polymerase III binds to 3’OH on the RNA primer and also to the
Guanine on the DNA template strand.
- Binding to Guanine opens up another active
site on DNA Polymerase III to bind CYTOSINE
(the binding partner for Guanine).
- Cytosine is H-Bonded to Guanine
- Cytosine – to – Adenine: Phosphodiester
bond via Dehydration Synthesis in 5’ to 3’
direction (uses energy from breaking
phosphate groups from nucleosides)
- DNA Polymerase III moves up
- Adds more nucleosides!
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DNA Polymerase III: A closer look
- Leading Strand: DNA Replication is
continuous from the 3’ end towards the
replication fork.
- Lagging Strand: DNA Replication is
discontinuous from the 3’ end near the
replication fork, and in a direction away from
the fork. We need MULTIPLE RNA primers.
- As the DNA is unzipped further, additional
RNA primers must be added to ”fill the
gaps”.
Fork is up here….
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The final stages
5. DNA Polymerase I: Removes the RNA primers and replaces them with DNA
nucleotides. (see next slide for the “problem”)
6. DNA Ligase: Seals the “nicks” i.e. creates phosphodiester bonds between
newly added nucleotides, called Okazaki Fragments present only on the
lagging strand.
7. DNA Polymerase I and III: “Proofread” the molecules looking for errors in
Nitrogenous Base Pairing. If errors are found, they replace the mistake with
the correct nucleotide. This is not perfect and “mutations” can still slip
through.
The “Problem”
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DNA Polymerase I: Removes the RNA
primers and replaces them with DNA
nucleotides….. But the LEADING STRAND
RNA primer at the 3’ end is removed but
NOT replaced with nucleotides!
Each time the cell divides, the DNA gets
shorter and shorter on the lagging strands.
These regions that are not being replicated
are called Telomeres.
The regions are non-coding (no genes,
sometimes called “junk DNA), but do
contribute to aging and death of cells after
multiple divisions eat into coding regions
of DNA.
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