DNA Replication and Repair (Text ref. 6.

4)

After Watson & Crick determined the double helix structure of DNA, scientists set out to determine the mechanism of
replication.
Two mechanisms were proposed: ______________________________________ and Conservative replication.

Meselson & Stahl: proved that DNA replication was semi-conservative:

• In 1958, Matthew Meselson & Franklin Stahl carried out an experiment that demonstrated that DNA replication
is semiconservative.
• Like Hershey & Chase, Meselson & Stahl used isotopes to label the parent DNA strands before replication. The
isotope they used was “heavy” nitrogen, 15N.
• E. coli bacteria were grown for 17 generations in a medium that contained 15N until all the cells had it
incorporated into their DNA.
• They then transferred the bacteria to a medium that contained only normal or “light” nitrogen, 14N and allowed
one or two rounds of replication.
• Any new DNA produced should have lighter 14N, thus making it less dense than the parent 15N DNA.
• To determine the density of the DNA, DNA was isolated and centrifuged.
• After centrifuging, the original heavy 15N DNA was in a single band.
• After one round of DNA replication, there was a single band of DNA at a density that was consistent with DNA
that would have one strand made of 15N and the other made of 14N.
• If replication had been conservative, two bands would have been seen, one containing the parental 15N DNA
and one containing only new 14N DNA.
• The results of centrifuging after two rounds of replication confirmed this conclusion.

DNA Replication: The Process

Three steps:
1. Strand Separation
2. Building Complementary Strands
3. Dealing with Errors during DNA Replication

Step 1: Strand Separation:

• To begin replication, the strands must be unwound from each other.
• Specific nucleotide sequences on the genome, called replication origins, act as starting points. (Note: there are
many replication origins on a eukaryote DNA strand.)
 • An enzyme called helicase binds to these origins and begins to unwind the two strands of DNA by breaking the
hydrogen bonds between the complementary base pairs.
• As the two strands separate, they form a Y-shaped structure known as a replication fork.
• The separation creates tension elsewhere in the strand. Enzymes called topoisomerases relieve this tension by
cutting one of the strands, and later rejoining it.
• The separated strand will have a tendency to want to re-attach or anneal. To prevent this, single-strand binding
proteins (SSBs) prevent annealing by attaching to the DNA strands to stabilize them and keep them separate.
• As replication forks proceed in opposite directions, the space between them is filled with newly replicated DNA
called a replication bubble.
• Each bubble expands along the length of the molecule until it meets and merges with another bubble,
eventually producing two separate daughter strands.
• The purpose of multiple replication origins is to reduce the time it takes for DNA to replicate (in humans, rate is
about 50 base pairs per second at each fork).

Step 2: Building Complementary Strands:

• During replication, 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 while moving along and ‘reading’
the template strand in its 3’ to 5’ direction. (Therefore, the new strand is always assembled in the 5’ to 3’
direction.)
• DNA polymerase builds the new strand of DNA using nucleoside triphosphates which are building blocks and
energy sources for replicating DNA.
• When the replication fork first opens, the DNA needs something to ‘add to’. RNA primase enzymes begin the
replication process by building a small complementary RNA segment on the strand at the beginning of the
replication fork. These short 10-60 ribonucleotide pieces are called RNA primers.
• DNA polymerase III then begins adding DNA nucleotides to the RNA primer. Since DNA polymerase only builds
in the 5’ to 3’ direction, the two new strands begin to be assembled in opposite directions.
• The leading strand does not require additional RNA primers, but the lagging does.
• On the lagging strand, the RNA primase must wait until enough of the fork has opened to attach another
primer.
• This causes a series of RNA primers and short DNA fragments called Okazaki fragments on the lagging strand.
• Each fragment extends in the 5’ to 3’ direction and attaches to the Okazaki fragment ahead of it.
• DNA polymerase I works to remove the RNA nucleotides one at a time and replace them with DNA nucleotides.
• The fragments are then joined together by an enzyme called DNA ligase.

Step 3: Dealing with Errors

• As DNA Polymerases assemble new DNA strands, they proofread and correct errors.
• Errors are usually base-pair mismatches.
• DNA Polymerase III cannot move forward if base pairs are mismatched. It will back up, replace the incorrect
base with the correct one and continue on.
• Errors after this may still occur but on average there may only be 1 error per one million base pairs.
• DNA Polymerase I moves along the DNA strand looking for distortions in shape (due to errors), and will excise
and repair.
• Similar repair mechanisms help to correct damage caused by chemicals and radiation (i.e. UV light).
• Without repair mechanisms, DNA would have many errors which could lead to loss of function and even cancer.