Lesson 3: DNA Replication Flashcards
DNA in Prokaryotes and Eukaryotes
Prokaryotes
Contain a single large double
stranded DNA molecule in
protein held in the cell’s nucleoid
region (pseudo – nucleus)
Contain one or more small
circular DNA molecules, called
plasmids, which float in the
cytoplasm
Eukaryotes
Contain double stranded DNA
formed in the nucleus of the cells
DNA binds with the protein
Histone to form Chromosomes
during cell division.
The Mechanism of DNA Replication
There were three possible mechanisms proposed for the replication of DNA:
Semi-conservative – the two strands of DNA separate and each serves
as a template for the synthesis of a new complementary strand
Conservative – the entire molecule serves as a template for a new
molecule
Dispersive – two strands break into units that are replicated and
reassembled, with the new molecule containing alternating segments.
Meselson and Stahl (1958)
Meselson and Stahl discovered the replication method by using two
different isotopes of Nitrogen and E. coli.
The E. coli was first grown in a medium containing “heavy” nitrogen
(15N). When extracted this DNA will be near the bottom of a solution of
CsCl.
DNA extracted from E.coli grown on normal nitrogen (14N) will be in the
less dense top of the solution of CsCl.
After time on the “heavy” nitrogen medium, the E.coli were transferred to a normal nitrogen medium
for one generation. Their DNA will contain half 15N and half 14N.
When placed in a CsCl solution there was one line halfway between the location of the 14N and
15N locations.
This observation eliminated the conservative model.
When grown for a second generation in 14N medium and placed in a CsCl tube, two bands were
observed.
One at the 14N/15N position and one at the 14N position.
This observation eliminated the dispersive model, leaving the semi-conservative model to be the
only one consistent with the observed results
DNA Replication in
Prokaryotes
Prokaryotic DNA is mostly circular molecules called
plasmids which float around the cytoplasm
The replication process of these circular molecules is
referred to as the “rolling wheel method”and has three
stages:
Stage 1: Initiation
A group of Initiation Enzymes called topoisomerases recognize a 100 – 200
base nucleotide sequence called the replication origin
The topoisomerases bind to the DNA at this site and separates the 2 strands
creating a replication bubble.
Stage 2: Elongation
DNA Polymerase III enters the bubble and binds to each parental strand of
DNA
DNA Polymerase III uses each strand of parental DNA as a template in order to
create a new complementary DNA strand one nucleotide at a time
DNA Polymerase replicates in the 5’ to 3’ direction
Replication forks move away from each other.
Stage 3: Termination
Once the replication forks meet the process is
over and the two molecules separate
DNA Replication in
Eukaryotes
Eukaryotic DNA is much larger than Prokaryotic DNA
Long, large double helical strands
More complicated process
Stage 1: Initiation
DNA Gyrase (a topoisomerase) recognizes
an origin of replication sequence on the
parent DNA (Eukaryotic DNA has many
origins of replication)
A section of the DNA unravels creating a
replication bubble
Helicase enters the replication bubble and
uncoils and separates the double helix
Single Stranded Binding Proteins (SSBP’s)
bind to the separated DNA strands to
prevent them from reattaching
Stage 2: Elongation
DNA Primase binds to each parent strand of DNA and
creates an RNA primer which is used as a starting
point for replication
DNA Polymerase III binds to both parental DNA
strands and will create 2 new DNA strands from the
existing parental DNA templates
DNA Polymerase III can only form a new strand in the
5’ to 3’ direction
The Problem: Parental DNA strands are anti – parallel
The replication process is DIFFERENT for each strand:
The 3’ – 5’ parental strand is called the Leading
Strand
The 5’ – 3’ parental strand is called the Lagging
Strand
Replication of the
Leading Strand
DNA Polymerase III creates a new complementary DNA
strand one nucleotide at a time.
This process is continuous and fairly fast since the
replication fork is moving in the same direction as the DNA
polymerase.
Replication of the
Lagging Strand
For this strand the DNA Polymerase is moving in the
opposite direction of the replication fork
The Lagging strand is replicated in pieces called Okazaki
Fragments
DNA Primase creates an RNA primer which is used as a starting point for the first
DNA fragment
DNA Polymerase III binds to the strand and forms a new strand from this point.
As more of the double helix is unwound, another RNA primer is formed by DNA
Primase and another DNA polymerase creates another fragment
DNA Polymerase I will then convert the RNA Primers into DNA
DNA Ligase then pieces the Okazaki Fragments together
DNA Polymerase III will then proofread each nucleotide and correct any mistakes in
replication
Stage 3: Termination
Once replication is complete the strands recoil into a double helix
Eventually the DNA polymerase III will reach an area of the DNA called the
Telomere
Telomeres are safe termination regions containing repeating TTAGGG
sequences.
The lifespan of the cell is dependent on the lifespan of the telomere regions
because every time DNA replicates the new daughter strand is a few
nucleotides shorter because the original RNA primer on the leading strand and
the last RNA primer on the lagging strand are not converted to DNA
