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BIO 184
Fall 2006
LECTURE 5
Lecture 5:
DNA Replication
Electron micrograph of bacteriophage lambda undergoing DNA replication inside its host
E. coli cell. http://www.biochem.wisc.edu/inman/empics/dna-prot.htm
I. DNA Replication is always “semi-conservative”
DNA replication is the process by which the genetic material is copied prior to
distrubution into daughter cells
 The original DNA strands are used as templates for the synthesis of new
strands
 It occurs very quickly, very accurately and at the appropriate time in the
life cycle of the cell
DNA replication relies on the complementarity of DNA strands
 The AT/GC rule or Chargaff’s rule
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The process can be summarized as follows:
o The two DNA strands in the parent DNA molecule come apart
o Each “parent strand” then serves as a template for the synthesis
of a new complementary strand
 The two newly-made strands = daughter strands
 The two original ones = parental strands
Parent DNA molecule
Daughter DNA molecules
“Daughter strands”
“Parental strands”
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This process is called semi-conservative because it conserves only half of the
original (parent) DNA molecule in the two daughter DNA molecules. One strand in
each daughter molecule is completely new.
In the late 1950s, three different mechanisms were proposed for the replication
of DNA
o Conservative model
 Both parental strands stay together after DNA replication and
one of the daughter molecules contains all new nucleotides
o Semiconservative model
 The double-stranded DNA contains one parental and one
daughter strand following replication
o Dispersive model
 Parental and daughter DNA are interspersed in both strands
following replication
See Figure 11.2, Brooker
In 1958, Matthew Meselson and Franklin Stahl devised a method to investigate
these models.
Their experiment can be summarized as follows:
o Grow E. coli in the presence of 15N (a heavy isotope of nitrogen) for
many generations
 The population of cells now has heavy-labeled DNA because the
DNA bases are rich in nitrogen
o Switch E. coli to medium containing only 14N (a light isotope of
nitrogen)
o Collect sample of cells after various times
o Analyze the density of the DNA by centrifugation using a CsCl
gradient
See Figure 11.3, Brooker
The actual data from the Mesleson-Stahl experiment is shown below.
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LECTURE 5
After ~ two generations, DNA is of
two types: “light” and “half-heavy”
This is consistent with only
the semi-conservative model
After one generation,
DNA is “half-heavy”
This is consistent with both
semi-conservative and
dispersive models
II. DNA Replication in Bacteria
DNA synthesis begins at a site termed the origin of replication (“ori”)
 Each bacterial chromosome has only one ori
 Synthesis of DNA proceeds bidirectionally around the bacterial
chromosome
 The “replication forks” eventually meet at the opposite side of the
bacterial chromosome
o This ends replication
See Figure 11.4, Brooker
Bacterial DNA replication has been studied most extensively in E. coli, the favorite
bacterial “model organism” of molecular geneticists.
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LECTURE 5
The ORI in E. coli is called “oriC”
 Three types of DNA sequences in oriC are functionally significant
 AT-rich region
 DnaA boxes
 GATC methylation sites
DNA replication is initiated by the binding of DnaA proteins to the DnaA box
sequences
 This binding stimulates the cooperative binding of an additional 20 to 40
DnaA proteins to form a large complex.
 This causes the DNA to twist and the puts torque on the nearby AT-rich
region to denature and form a replication bubble
o AT base pairs are held together by only 2 H bonds
o CG base pairs are held together by 3 H bonds
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LECTURE 5
o Therefore, AT-rich regions of DNA denature more easily than CG-rich
regions of DNA
In the next step, DnaB (also called helicase) binds to each strand of the separated
double helix. It’s job is to move along the DNA, progressively expand the
replication bubble in both directions.
Travels along
the DNA
strand in the 5’
to 3’ direction,
using energy
from ATP
As the helicases move on each strand in opposite directions, two replication forks
are created. These forks move progressively farther and farther in each direction
as the bubble widens.
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BIO 184
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LECTURE 5

DNA helicase separates the two DNA strands by breaking the hydrogen
bonds between them
o This generates positive supercoiling ahead of each replication fork so
another enzyme, topoisomerase, travels ahead of the helicase and
alleviates these supercoils

Single-strand binding proteins (SSBPs) are also needed to bind to the
separated DNA strands and keep them apart
o Otherwise, the strands would simply reanneal

After the helicase, gyrase, and SSBPs are in place, short (10 to 12
nucleotides) RNA primers are synthesized by DNA primase
o These short RNA strands start, or prime, DNA synthesis because
DNA polymerase, the enzyme that copies DNA, cannot start a new
strand on its own
o The RNA primers are later removed and replaced with DNA
Breaks the hydrogen bonds
between the two strands
Keep the parental
strands apart
Alleviates
supercoiling
Synthesizes an
RNA primer
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LECTURE 5
III. DNA Polymerases
DNA polymerases are the enzymes that catalyze the attachment of nucleotides to
make new DNA

In E. coli there are five proteins with polymerase activity
o DNA pol I
 Composed of a single polypeptide
 Removes the RNA primers and replaces them with DNA during
DNA replication
o DNA pol III
 Composed of 10 different subunits (Table 11.2)
 The a subunit synthesizes DNA
 The other 9 fulfill other functions
 The complex of all 10 is referred to as the “DNA pol III
holoenzyme”
 Is responsible for most of the DNA replication process
o DNA pol II, IV and V
 Specialized DNA polymerases that replicate short areas of
DNA for the purposes of genome repair

The numbering of these polymerases was done in the order they were
discovered
Bacterial DNA polymerases may vary in their subunit composition. However, they
have the same type of catalytic subunit.
Structure
resembles a human
right hand:
Template DNA thread
through the palm;
Thumb and fingers
wrapped around the
DNA
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LECTURE 5
All DNA polymerases, whether bacterial or eukaryotic, share 2 very important
limitations:
1.
They cannot initiate DNA synthesis on their own. They require that an
RNA primer be laid down on the DNA first by DNA primase.
2.
They can only “grow” a new DNA chain in the 5’ to 3’ direction.
It is not fully understood why all DNA polymerases have these limitations. As will
be demonstrated below, DNA replication would be much simpler if they did not!
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LECTURE 5
Because DNA polymerase can only synthesize a new strand 5’ to 3’, the two new
daughter strands are synthesized in different ways:
o Leading strand
 One RNA primer is made at the origin
 DNA pol III attaches nucleotides in a 5’ to 3’ direction as it
slides toward the replication fork
o Lagging strand
 Synthesis is also in the 5’ to 3’ direction
 However it occurs away from the replication fork
 Many RNA primers are required
 DNA pol III uses the RNA primers to synthesize small DNA
fragments (1000 to 2000 nucleotides each)
 These are termed Okazaki fragments after their
discoverers
 DNA pol I removes the RNA primers and fills the
resulting gap with DNA
 After the gap is filled, a covalent bond is still missing so
DNA ligase must create this bond
Can be synthesized continuously in the
5’ to 3’ direction
Must be synthesized
discontinuously to
maintain 5’ to 3’
synthesis
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LECTURE 5
Note that if DNA polymerase was able to synthesize a new strand in either
direction (5’ to 3’ or 3’ to 5’), lagging strand synthesis and Okasaki fragments would
not be needed.
The process can also be visualized in 3-D as follows:
IV. The Synthesis Reaction
DNA polymerases catalyze a phosphodiester bond between the innermost
phosphate group of the incoming deoxynucleoside triphosphate and the
3’-OH of the sugar of the previous deoxynucleotide.
o
In the process, the last two phosphates of the incoming nucleotide are
released in the form of pyrophosphate (PPi)
See Figure 11.10, Brooker
In E. coli, DNA pol III stays on the DNA template long enough to polymerize up to
50,000 nucleotides at a rate of ~ 750 nucleotides per second!
V. Proofreading
DNA replication exhibits a high degree of fidelity.

Mistakes during the process are extremely rare
 In E. coli, DNA pol III makes only one mistake per 108 bases
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There are several reasons why fidelity is high:
1. Instability of mismatched pairs
o Complementary base pairs have much higher stability than mismatched
pairs
o This feature only accounts for part of the fidelity
o It has an error rate of 1 per 1,000 nucleotides
2. Configuration of the DNA polymerase active site
o DNA polymerase is unlikely to catalyze bond formation between
mismatched pairs
o This induced-fit phenomenon decreases the error rate to a range of 1
in 100,000 to 1 million
3. Proofreading function of DNA polymerase
o DNA polymerases can identify a mismatched nucleotide and remove it
from the daughter strand
o The enzyme uses its 3’ to 5’ exonuclease activity to remove the
incorrect nucleotide
o It then changes direction and resumes DNA synthesis in the 5’ to 3’
direction
VI. Termination of Replication in Bacteria
DNA replication ends when oppositely advancing forks meet (remember that the
chromosome is circular).
 DNA replication often results in two intertwined molecules called catenanes
 Catenanes and are separated prior to cell division
Replication
Decatenization
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LECTURE 5
VII. DNA Replication in Eukaryotes
Eukaryotic DNA replication is not as well understood as bacterial replication.


The two processes do have extensive similarities,
Many of the bacterial enzymes described above have also been found in
eukaryotes

Nevertheless, DNA replication in eukaryotes is more complex due to:
o Large linear chromosomes
o Multiple origins of replication per chromosome
o Tight packaging of the DNA around proteins
See Figure 11.20, Brooker
Linear eukaryotic chromosomes also have telomeres at both ends
 The term telomere refers to the complex of repetitive DNA sequences
found at the terminal ends of eukaryotic chromosomes as well as the
proteins that recognize this sequence and bind the DNA there.
Telomeric sequences consist of
 Moderately repetitive tandem arrays
 3’ overhang that is 12-16 nucleotides long that results from the loss of the
RNA primer at the 5’ end of each strand that cannot be replaced.
http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/T/Telomeres.html
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LECTURE 5
See Figure 11.24, Brooker
Therefore if this problem is not solved:
 The linear chromosome becomes progressively shorter with each round of
DNA replication
 Indeed, some cells solve this problem by adding DNA sequences to the ends
of telomeres following replication
o This requires a specialized mechanism catalyzed by the enzyme
telomerase
o All single-celled eukaryotes have active telomerase enzyme because if
they didn’t successive generations of the organism would have
shortened telomeres
 Eventually, this would result in the loss of important genes and
the death of the species
However, most somatic cells in multicellular organisms do not express telomerase
and the telomeres shorten every time the cells replicate.
o Most human somatic cells can only replicate about 30 times before
their telomeres are so shortened that the cell dies
 This sets an upper limit on the life span of the organism
Telomerase is active in the germ line cells, maintaining telomere length from one
generation to the next.
Telomerase is also often abnormally activated in cancer cells. This is why tumors
don’t eventually replicate themselves to death. Once a tumor cell has activated
telomerase, it is immortalized.
Immortalized somatic cells are extremely dangerous in multicellular organisms. If a
cell suffers a mutation in a gene controlling the cell cycle, the cell can begin to
replicate much faster than the surrounding cells.
 Normally, such cells will die off when their telomeres get too short
 Immortalized cells will continue to cycle and the tissue will grow
 The result can be a cancerous tumor that is life-threatening to the
organism
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