DNA Replication and Repair

DNA Replication and Repair

• DNA replicates itself

• Millions of nucleotides in
length
• Replication must occur very
quickly and with minimal
errors
• Watson and Crick’s 3-D model
of DNA gave scientists a place
to begin
DNA Replication is Semi-Conservative

• Semi-conservative replication involves

separating the two parent strands and building a
new, complementary replacement strand for each
• New molecule consists of one parent strand and
one new strand
• Conservative replication on the other hand would
mean that the original parent strand would stay
together
• Meselson and Stahl (1958) used isotopes and e.
coli bacteria to label parent DNA strands before
replication (Pg. 282. Fig. 1)
• Each single strand of parent DNA has been used
as a template for a new complementary DNA
strand
DNA Replication: The Process

• There are three main

steps to DNA
replication:
1. The parental strands of
DNA separate
2. The complementary
DNA strands are
assembled
3. The new strands are
proofread and repaired
Step 1: Strand Separation

• The DNA must first be unwound from

each other
• Specific nucleotide sequences act as
replication origins (starting points)
• There can be many different replication
origins per strand due to the length
• Enzyme called Helicase binds to the
replication origins and begins to unwind
strands by breaking the hydrogen bonds
between the base pairs
• Separation of strands creates a Y-shaped
replication fork
Step 1: Strand Separation
• A class of enzymes called topoisomerases
relieves the tension caused by the unwinding
of parent DNA
• They cleave one or two of the DNA strands,
allowing them to untwist, and then rejoin the
stands
• Single strand binding proteins (SSBs) prevent
annealing (strands coming back together
before they should)
• Helicase can separate in both directions from
replication origin creating replication bubbles
of newly formed DNA
• Numerous replication bubbles allows for new
DNA to be formed at 50 base pairs per second
Step 2: Building Complementary Strands
• New nucleotides are joined by a group of enzymes called DNA polymerases
• DNA polymerases add nucleotides to the 3’ end of a new developing strand reading the template
strand 5’ – 3’
• Therefore, new strand can only be assembled in the 5’ to 3’ direction
• DNA builds new strand using nucleoside triphosphates (building block and energy source)
• Energy to drive the DNA synthesis is provided by hydrolysis of phosphate groups
Step 2: Building Complementary Strands

• RNA primase builds a small

complementary RNA segment at the
beginning of the replication fork
• DNA polymerase III begins adding DNA
nucleotides to the RNA primer in the 3’ – 5’
direction
• Therefore, two new strands will begin to
be assembled in opposite directions
• As the replication fork continues to open,
The DNA polymerase III building the
strand towards the fork can continuously
add nucleotides
• This is called the leading strand (new
strand heading towards replication fork)
 Step 2: Building Complementary Strands

• On the other template strand DNA polymerase is

moving away from the replication fork (Lagging
strand)
• When enough fork has opened RNA primase attaches
RNA primer to the template (parent) strand
• Therefore, the lagging strand gets built in sections
called Okazaki Fragments
• Lagging strand is discontinuous (it needs multiple
RNA primers and Okazaki fragments to be completed
• Another polymerase, DNA polymerase I removes
RNA nucleotides and replaces with DNA
• DNA ligase catalyzes the final linkage between the
DNA segment and the Okazaki fragment
Step 3: Dealing with Errors during DNA

• DNA polymerase enzymes proofread and

correct errors as they build new strands
• Average of 1 error in every million base
pairs
• Once, done, DNA polymerase I and II
read the new strands for errors
• DNA polymerase fixes the left over errors
and DNA ligase fuses the strand together
• These are constantly working to correct
damage to DNA from environmental
factors (chemicals and radiation)
 DNA Replication in Prokaryotes and Eukaryotes
• Most DNA knowledge has come from
bacteria studies
• Process is very similar in eukaryotes
• Prokaryote genomes can be different
shape (circular) or smaller than eukaryotes
• Only one replication origin and bubble in
eukaryotes
• Understanding DNA replication could
have significant implications for growth,
aging, cloning, tissue regeneration, cancer,
and biotechnology