Mrs.

OFELIA SOLANO SALUDAR

Department of Natural Sciences
University of St. La Salle
Bacolod City
 Tommy was a full-term baby but weighed only 4.5 pounds (2
kg) at birth. At about 9 months of age, an unusual and persistent
rash appeared on his face, and he frequently caught colds and
infections. The illnesses caused no serious problems; so his
parents were not concerned.
Throughout childhood, Tommy remained small; by age 18, he
was only 4 feet 6 inches (137 cm) in height. Tommy’s first major
health problem arose shortly after he turned 22—he was
diagnosed with intestinal cancer. The tumor was surgically
removed but additional, unrelated tumors appeared
spontaneously over the next 10 years.
Their appearance startled Tommy’s doctors; the chance of
multiple, independent cancers arising in the same person is
generally remote.
 The propensity of Tommy’s cells to become cancerous hinted
at a high mutation rate in his genes. Indeed, when pathologists
studied Tommy’s chromosomes, they observed a wide range of
abnormalities. Tommy had inherited BLOOM SYNDROME.
Bloom syndrome is a rare autosomal recessive condition
characterized by short stature, a facial rash induced by sun
exposure, a small narrow head, and a predisposition to cancers
of all types.
The disorder is extremely rare; only several hundred cases
have been reported worldwide. Cells from persons with Bloom
syndrome exhibit excessive mutations in all genes, and numerous
gaps and breaks occur in chromosomes that lead to extensive
genetic exchange in cell division. Rates of DNA synthesis are
retarded.
 The characteristics of Bloom syndrome suggest that
its underlying cause is a defect in DNA replication. In
1995, researchers at the New York Blood Center traced
Bloom syndrome to a gene on chromosome 15 that
encodes an enzyme called DNA helicase. A variety of
helicase enzymes are responsible for unwinding double-
stranded DNA during replication and repair.
The cells of a person with Bloom syndrome carry two
mutated copies of the gene and possess little or no
activity for a particular helicase. Normal DNA
replication is disrupted, leading to chromosome breaks
and numerous mutations. The genetic damage resulting
from faulty DNA replication leads to tumors.
It is not yet clear whether the basic defect in DNA
synthesis is associated with replication or DNA repair or
both.
 To understand Tommy’s case, we need to answer the
following questions:

 What models of DNA replication exist among life forms?

 Where is the origin of replication in the DNA strand?
 What is the direction of replication at this site?
 How does the chain grow in length?
 How does the chain terminate?
 What is the enzymology behind DNA replication?
 Are there other protein factors that must be present?
 What is the role of DNA replication in the expression of
disease?
 MODELS OF DNA REPLICATION

These models may differ with respect to the initiation

and progression of replication, but all produce new DNA
molecules by semi-conservative replication.
THETA REPLICATION: E. coli
 ROLLING CIRCLE:
Viruses and F factor of E. Coli
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FUNDAMENTAL RULES OF DNA REPLICATION
1. Replication is semi-conservative
Meselson and Stahl convincingly demonstrated that each E. coli
DNA strand serves as a template for the synthesis of a new
DNA molecule.
 2. Replication
begins at an
origin- the
replication fork
 3. DNA replication is bi-directional, and
proceeds in a 5’-3’ direction

DNA synthesis takes place simultaneously but in opposite

directions on the 2 template strands.
4. DNA Replication is Semi-discontinuous

Replication fork

lagging strand Replication fork

Leading strand
The polarity of
DNA synthesis
creates an
asymmetry
between the
leading strand
and the
lagging strand
at the
replication
fork
 Requirements of Replication

Although the process of replication includes many

components, they can be combined into three major
groups:
1. a template consisting of single-stranded DNA,
2. raw materials (substrates) to be assembled into a new
nucleotide strand, and
3. enzymes and other proteins that “read” the template
and assemble the substrates into a DNA molecule.
New DNA is synthesized from deoxyribonucleotide triphosphates (dNTPs).
Since the 5’ end does not get added to and the 3’ end repeatedly does, the DNA
strand is said to grow in a 5’- 3’ manner.
Components required for replication
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DNA Polymerases in E. coli

DNA Polymerase III

 Topoisomerase

Protein complexes of the

replication fork
 DNA helicase
unwinds
the DNA duplex
ahead of DNA
polymerase
creating single
stranded DNA that
can be used
as a template
ssDNA binding proteins bind to the sugar phosphate backbone leaving the
bases exposed for DNA polymerase. The binding of SSB to newly formed
ssDNA prevents reassociation of the single strands and “iron out” the
unwound DNA.
 Since DNA polymerase
requires a template and a free
3’ OH group to add
nucleotides on to, RNA
primers are required to initiate
DNA polymerization.
Primase, an enzyme which is
part of a large complex of
proteins called the
primosome, synthesizes a
small stretch of RNA (the
primer) of 3-10 nucleotide in
length, which will act as a
starting site for the DNA
polymerase.
 DNA polymerase
falls off the DNA
easily. A “sliding clamp”
is required to keep DNA
polymerase on and allow
duplication of long
stretches of DNA
 A “clamp
loader:”
complex is
required
to get the clamp
onto the DNA
 Ahead of the
replication fork
the DNA
becomes
supercoiled

The supercoiling needs to be

relieved or tension would
build up (like coiling as
spring) and block fork
progression.
 Supercoiling is relieved by the action of
Topoisomerases.

1. Type I topoisomerases:
 Make nicks in one DNA strands
 Can relieve supercoiling
2. Type II topoisomersases or DNA gyrase
 Make nicks in both DNA strands (double strand
break)
 Can relieve supercoiling and untangle linked DNA
helices
 Both types of enzyme form covalent intermediates
with the DNA
Type I Topoisomerase
Type II Topoisomerase
 Topoisomerases as drug targets
1. Dividing cells require greater topoisomerase
activity due to increased DNA synthesis
2. Topoisomerase inhibitors which act by stablilizing
the DNA-topoisomerase complex are used as
chemotherapeutic agents:
 camptothecin -Topo I inhibitor doxorubicin --
Topo II inhibitor
 Some antibiotics are inhibitors of the bacterial-
specific topoisomerase DNA gyrase: e.g.
ciprofloxacin
D
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Replication is extremely accurate, with less than one error per billion
nucleotides. This accuracy results from the processes of nucleotide
selection, proofreading, and mismatch repair.
 DNA mismatch repair corrects errors made during DNA
replication.(A) If uncorrected, the mismatch will lead to a permanent
mutation in one of the two DNA molecules produced by the next round of
DNA replication. (B) If the mismatch is “repaired” using the newly
synthesized DNA strand as the template, both DNA molecules produced by
the next round of DNA replication will contain a mutation. (C) If the
mismatch is corrected using the original template (old) strand as the template,
the possibility of a mutation is eliminated. The scheme shown in (C) is used
by cells to repair mismatches.
 Chemical modifications of nucleotides, if left unrepaired, produce mutations.
(A) Deamination of cytosine, if uncorrected, results in the substitution of one base for another when the
DNA is replicated. Deamination of cytosine produces uracil. Uracil differs from cytosine in its base-pairing
properties and preferentially base-pairs with adenine. The DNA replication machinery therefore inserts an
adenine when it encounters a U on the template strand. (B) Depurination, if uncorrected, can lead to the loss
of a nucleotide pair. When the replication machinery encounters a missing purine on the template strand, it
can skip to the next complete nucleotide, thus producing a nucleotide deletion in the newly synthesized
strand. In other cases, the replication machinery places an incorrect nucleotide across from the missing base,
again resulting in a mutation.
 THE EUKARYOTIC REPLICON
Time for DNA replication is limited in the S phase of eukaryotes
(6-8 hrs in mammals. Such RFs move only about 1/10th of the
prokaryotic forks, and chromosomes can be in excess of 108 bp.
Completion of replication at the allotted time requires multiple
RFs called replicons. The Origin Recognition Complex (ORC)
is a complex of 6 ATPases which is the functional equivalent of
DnaA.
EUKARYOTIC DNA POLYMERASES
 How does a
linear
chromosome
close
replication at
its two ends?
 As DNA synthesis
requires a RNA primer
that will eventually be
digested away, standard
DNA replication would
result in linear
chromosomes that would
shrink with every round
of replication. This is
resolved in bacteria by
the circular genome
which does not have an
end. In linear
chromosomes, the
telomere solves the DNA
end replication problem.
 Telomeres have highly
repeated DNA sequences
5'-TTAGGG-3'.
 Human chromosomes
have between 100 and
1500 copies of this
sequence.
 Telomerase, a special
DNA polymerase, can
add additional copies of
the 5'-TTAGGG-3' to the
end of a chromosome.
 The telomerase enzyme
is actually a complex
containing protein and
RNA (a "ribozyme").
 The RNA portion has a 5'-CCCTAA-3' region that acts as a template
for adding the DNA repeat to the chromosome ends.
 The telomerase enzyme is found mostly in the germ cells of
multicellular organisms.
 In somatic cells, the absence of telomerase results in shorter
chromosomal ends with each division and may be the limiting factor
in an organism's life span.
TELOMERASE
AND DISEASE
 Errors of DNA Replication and Disease
 Origins or replication are strictly controlled so that
they “fire” only once per cell cycle
 Errors lead to over-replication of specific
chromosomal regions = gene amplification
 This is commonly seen in cancer cells and can be
an important prognostic indicator.
 It can also contribute to acquired drug resistance,
e.g. Methotrexate induces amplification of the
dihydrofolate reductase locus.
 The rate of misincorporation of bases by DNA polymerase is
extremely low, however repeated sequences can cause
problems.
 In particular, trinucleotide repeats cause difficulties which can
lead to expansion of these sequences.
 Depending where the repeat is located, expansion of the
sequence can have severe effects on the expression of a gene or
the function of a protein.

Looping out of repeats

before replication.
Several inherited diseases are associated with expansion of
trinucleotide repeat sequences.

Very different disorders, but they share the characteristic of

becoming more severe in succeeding generations due to
progressive expansion of the repeats
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