REPLICATION OF DNA

OBJECTIVES
• At the end of this lecture Students must be
able to
• 1. List the enzymes involved in DNA replication
and their functions
• 2. describe the Okazaki fragments
• 3. List the steps involved in Replication
• 4. list Enzymes involved in the DNA repair
• 5. Clinical application.
 Replication Facts

• DNA has to be copied

before a cell divides
• DNA is copied during the S
or synthesis phase of
interphase
• New cells will need identical
DNA strands

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Synthesis Phase (S phase)
• S phase during interphase of the
cell cycle
• Nucleus of eukaryotes
S
DNA replication takes phase
place in the S phase.

G1 interphase G2

Mitosis
-prophase
-metaphase
-anaphase
-telophase
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 DNA Replication is “Semi-conservative”

• Each 2-stranded
daughter molecule is
only half new
• One original strand
was used as a template
to make the new strand
*
Origin of
Replication
• Specific DNA sequences
• Attract initiator proteins
• Easy to unwind/open
• Fewer bonds A-T
• “AT rich” sequences
• Easy to open

Wikipedia/Public Domain
 EUKARYOTIC
• A pre-replication complex (pre-RC) is a protein
complex that forms at the origin of
replication during the initiation step of DNA
replication.
• Composed of ORC and homohexamer of the mini
chromosome maintenance (MCM) protein.
• Origin Recognition Complex (ORC) is a multi-
subunit DNA binding complex (6 subunits) that
binds in all eukaryotes in an ATP-dependent
manner to origins of replication.
 DNA Replication
• As the 2 DNA strands open at
the origin, Replication Bubbles
form
• Prokaryotes (bacteria) have a
single bubble
• Eukaryotic chromosomes have
MANY bubbles
Bubbles Bubbles

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 DNA replication
• The parent molecule unwinds, and two new
daughter strands are built based on base-
pairing rules

T
A T A T A A T A T A T
C G C G C G C G C G C G
T A T A T A T A T A T A
A T A T A T A T A T A T
G C G C G C C G C G C
G

(a) The parent molecule has two
complementary strands of DNA.
Each base is paired by hydrogen
bonding with its specific partner,
A with T and G with C.
(b) The first step in replication is
separation of the two DNA
strands.
(c) Each parental strand now (d) The nucleotides are connected
serves as a template that to form the sugar-phosphate
determines the order of backbones of the new strands.
nucleotides along a new, Each “daughter” DNA
complementary strand. molecule consists of one parental
strand and one new strand.
• DNA replication occurs in the nucleus of a cell
and during S phase of cell cycle.
• The overall process of DNA replication
requires the synthesis of both DNA and RNA.
• The enzyme involved is called DNA
polymerase and RNA polymerase respectively.
Primers
• DNA polymerase cannot initiate replication
• Primers: short nucleotide sequences
• Formed at point of initiation of new chain
• Required by DNA polymerase to function
Primers
• DNA Primase: Makes primers
• Primers contain RNA
• Ribonucleotides (not deoxy-ribonucleotides)
• Uracil instead of thymine
• Eventually removed and replaced with DNA

Ribonucleotide Deoxyribonucleotide
DNA Polymerases
• Bacteria (prokaryotes)
• DNA polymerase I-IV
• Polymerase III: Major DNA polymerase
• Polymerase I: Removes RNA primers
• Eukaryotes
• DNA polymerase α, β, γ, δ, and ε
• Polymerase γ: located in mitochondria
 Enzymes in DNA replication

Helicase unwinds Binding proteins Primase adds

parental double helix stabilize separate short primer
strands to template strand

DNA polymerase III DNA polymerase I Ligase joins Okazaki

binds nucleotides (Exonuclease) removes fragments and seals
to form new strands RNA primer and inserts other nicks in sugar-
the correct bases phosphate backbone
 Replication
3’
3’ 5’
5’
3’

5’ 3’
5’

Helicase protein binds to DNA sequences called

origins and unwinds DNA strands.

Binding proteins prevent single strands from rewinding.

Primase protein makes a short segment of RNA
complementary to the DNA, a primer.
DNA Replication
• Helicase
• Unwinds/opens double helix
• Hydrolyzes ATP
• Single strand binding proteins
• Assist helicase
• Stabilize and straighten single strands of DNA
 Replication
Overall direction
3’
of replication
3’ 5’
5’

3’

5’ 3’
5’

DNA polymerase enzyme adds DNA nucleotides

to the RNA primer.
DNA Replication Directionality
• Always occurs in 5’ to 3’ direction
• Nucleotides added to 3’ end of growing strand
 Replication
Overall direction
3’
of replication
3’ 5’

5’
3’

5’ 3’
5’

DNA polymerase enzyme adds DNA nucleotides

to the RNA primer.

DNA polymerase proofreads bases added and

replaces incorrect nucleotides.
 Replication
Overall direction
3’
of replication
3’ 5’

5’
3’

5’ 3’
5’

Leading strand synthesis continues in a

5’ to 3’ direction.
 Replication
Overall direction
3’
of replication
3’ 5’

5’
Okazaki fragment
3’

5’ 3’ 5’ 3’
5’

Leading strand synthesis continues in a

5’ to 3’ direction.

Discontinuous synthesis produces 5’ to 3’ DNA

segments called Okazaki fragments.
 Replication
Overall direction
3’
of replication
3’ 5’

5’
Okazaki fragment
3’

5’ 3’ 5’ 3’
5’

Leading strand synthesis continues in a

5’ to 3’ direction.

Discontinuous synthesis produces 5’ to 3’ DNA

segments called Okazaki fragments.
 Replication

3’
3’ 5’

5’
3’
5’ 3’ 5’ 3’5’ 3’
5’

Leading strand synthesis continues in a

5’ to 3’ direction.

Discontinuous synthesis produces 5’ to 3’ DNA

segments called Okazaki fragments.
 Replication

3’
3’ 5’

5’
3’
5’ 3’5’ 3’5’ 3’
5’

Leading strand synthesis continues in a

5’ to 3’ direction.

Discontinuous synthesis produces 5’ to 3’ DNA

segments called Okazaki fragments.
 Replication

3’
3’ 5’

5’
3’
5’ 3’5’ 3’5’ 3’
5’

Exonuclease activity of DNA polymerase I removes RNA primers.

 Replication

3’
3’

5’
3’
5’ 3’5’ 3’
5’

Polymerase activity of DNA polymerase I fills the gaps.

Ligase forms bonds between sugar-phosphate backbone.
Replication Fork Overview
supercoils
 DNA Replication
• Enzyme Topoisomerase attaches
to the 2 forks of the bubble to
relieve stress on the DNA
molecule as it separates
Enzyme Enzyme

DNA

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Topoisomerase
• Prevent DNA tangling
• Break DNA then reseal to relieve tension/twists
• Topoisomerase I
• Break single strands of DNA then reseal
• Topoisomerase II
• Break double strands then reseal

Topoisomerases
Topoisomerase
Clinical Correlations

• Quinolone antibiotics
• Prokaryotic topoisomerases
• Chemotherapy agents
• Eukaryotic toposiomerases
• Etoposide/teniposide
• Irinotecan, topotecan
• Anthracyclines
• Telomeres are repetiitive sequence at the ends
of linear 3’ DNA molecules in eukayotic
chromosome. With each round of replication in
most normal cells, the telomeres are shortened
and DNA polymerase cannot complete synthesis
of 5’ end of each strand. This contributes to
aging of cells, because eventually telomers
become so short the chromosomes cannot
function properly and d cells die.
• TELOMERASE: Is the enzyme used to maintain
the telomeres. And it is able to replace
telomere sequence that would otherwise be
lost during replication. Normally telomerase
activity is present only in embryonic cells,
germ cells, and stem cells but not adult
somatic cells.
• WHY?
• Telomerase activity is inappropriately present
in cancer cells. Preventing the telomeres from
being shortened and contributing to
immortality of the cancer cells.
 The Mechanism of DNA Replication
• Many proteins assist in DNA replication

• DNA helicases unwind the double helix, the

template strands are stabilized by other proteins

• Single-stranded DNA binding proteins make the

template available

• RNA primase catalyzes the synthesis of short

RNA primers, to which nucleotides are added.

• DNA polymerase III extends the strand in the

5’-to-3’ direction

• DNA polymerase I degrades the RNA primer

and replaces it with DNA

• DNA ligase joins the DNA fragments into a

continuous daughter strand
 Replication of Strands
Replication Point of Origin
Fork

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 Application
• Quinolones are a family of drugs that block the
action of topoisomerases.
• Nalidixic acid kills bacteria by inhibiting DNA
gyrase.
• Inhibitors of eukaryotic topoisomerase II
(etoposide, teniposide) are becoming useful as
anticancer agents.
 DNA REPAIR
• The structure of DNA can be damaged in a
number of ways through exposure to chemicals
or radiation (UV, gamma, X-rays). Multiple repair
systems have evolved, allowing cells maintain the
sequence stability of their genome.
• If cells are allowed to replicate their DNA using
damaged template, there is a high risk of
introducing stable mutations into the new DNA.
Types of DNA Damage
• Depurination
• Occurs spontaneously thousands of times per day
• Results in loss of purine bases (guanine and adenine)
• Deamination
• Occurs spontaneously hundreds of times per day
• Base loses amine group (cytosine)

Adenine Guanine
 Proofreading and Repairing DNA
• DNA polymerases 1 A thymine dimer

proofread newly made distorts the DNA molecule.

2 A nuclease enzyme cuts

DNA, replacing any the damaged DNA strand

at two points and the
damaged section is
incorrect nucleotides removed.
Nuclease
• In mismatch repair of
DNA, repair enzymes DNA
polymerase
3 Repair synthesis by
a DNA polymerase

correct errors in base fills in the missing

nucleotides.

pairing DNA
ligase 4 DNA ligase seals the
• In nucleotide excision Free end of the new DNA
To the old DNA, making the
DNA repair nucleases cut strand complete.

out and replace damaged

stretches of DNA
 A failure to repair DNA produces
a mutation.
• 1. Excision Repair, in which the damaged base or bases are
removed and then replaced with the correct ones in a localized
burst of DNA synthesis. There are three modes of excision
repair, each of which employs specialized sets of enzymes.
• 2. Base Excision Repair (BER). DNA glycosylase.
• Removal of the damaged base (estimated to occur some
20,000 times a day in each cell in our body!) by a DNA
glycosylase. DP Beta.
3. Nucleotide Excision Repair (NER). Transcription Factor
IIH, TFIIH.
• Designated polymerase delta and epsilon.
• 3. Mismatch Repair (MMR)
• Recognition of a mismatch requires several
different proteins including one encoded
by MSH2.
• Cutting the mismatch out also requires several
proteins, including one encoded by MLH1
• Thus any defect in DNA repairs carries and
increase risk of cancer. Most DNA repairs
occur in G1 phase of cell cycle.
• Mismatch repair occurs in G2 phase to correct
replication errors.
Base Excision Repair
• Pathway for damaged DNA repair
• Recognize specific base errors
• Deaminated bases, oxidized bases, open rings
• Numerous variations/enzymes used by cells
• Functions throughout the cell cycle (all phases)
Base Excision Repair
• DNA glycosylases
• Several different enzymes
• Remove damaged bases
Glycosidic
• Creates a baseless nucleotide Bond
• “Apurinic” or “apyrimidic” nucleotide

A T C G A C G
T A G C T A G C
Base Excision Repair
• AP endonuclease
• Recognizes nucleotides without a base
• Attacks 5’ phosphate end of DNA strand
• “Nicks” damaged DNA upstream of AP site
• Create a 3'-OH end adjacent to the AP site
• AP lyase
• Some DNA glycosylases also possess AP lyase activity
• Attack 3’ hydroxyl end of ribose sugar

A C G A C G

T A G C T A G C
Base Excision Repair
AP
Endonuclease

AP
Lyase

Wikipedia/Public Domain
Base Excision Repair
• DNA polymerase
• Adds new nucleotide (complementary to opposite base)
• Extends 3'-OH terminus
• DNA ligase seals strand

A C G A T C G

T A G C T A G C
Nucleotide Excision Repair
• Removes “bulky” DNA damage
• Multiple bases
• Often pyrimidine dimers
• Commonly caused by UV radiation (sunlight)
• G1 phase (prior to DNA synthesis)
• Endonucleases removed multiple nucleotides
• DNA polymerase and ligase fill gap

Public Domain
Nucleotide Excision Repair
Cyclobutane
Pyrimidine
Dimer

Thymidine Thymidine

Wikipedia/Public Domain
Xeroderma Pigmentosum
• Defective nucleotide excision repair
• Extreme sensitivity to UV rays from sunlight
• Signs appear in infancy or early childhood
• Very easy sunburning
• Freckling of skin
• Dry skin (xeroderma)
• Changes in skin pigmentation
• Very high risk of skin cancer
• May develop in childhood

James Halpern, Bryan Hopping and Joshua M Brostoff

Mismatch Repair
• Identifies incorrectly placed bases/nucleotides
• Insertions, deletions, incorrect matches
• Occurs when proofreading misses errors
• No damage to base – not recognized by repair systems
• Occurs in S/G2 phase (after DNA synthesis)
• Newly synthesized strand compared to template
• Nucleotide errors removed and resealed
Mismatch Repair
• Important for microsatellite stability
• DNA has many repeating segments
• “Microsatellites”

A AAACCC GGTT
C CCCAAA TTGG
Mismatch Repair
• DNA slippage can occur at repeats
• Results in a mismatch
• Repaired by MMR systems
• Result: number of repeats (microsatellites) stable
Mismatch Repair
• Microsatellite instability
• Results if MMR systems deficient
• Seen in cancers cells (colon cancer)
HNPCC
Hereditary Non-Polyposis Colorectal Cancer/Lynch Syndrome

• Germline mutation of DNA mismatch repair enzymes

• About 90% due to MLH1 and MSH2 mutations
• Leads to colon cancer via microsatellite instability
• About 80% lifetime risk
• Hallmark: cancer cells with microsatellite instability
Double Strand Damage
• Commonly result from exogenous sources
• Ionizing radiation
• Caused by radiation therapy (cancer)
Homologous End
Joining
• Homology = similar structure
• HEJ = uses sister chromosome template

Sister
Chromosome
Non-Homologous End
Joining
• Uses many proteins to re-join broken ends
• DNA pol λ and μ re-extend the ends
• Many other enzymes
• No template used (non-homologous)
• Highly error-prone

NHEJ
Double Strand Break
Fanconi Anemia
• Inherited aplastic anemia
• More than 13 genetic abnormalities identified
• Many involve DNA repair enzymes
• Hypersensitivity to DNA damage
• Cells vulnerable to DNA strand cross-links
• Also impaired homologous recombination
Ataxia Telangiectasia
• Defective Nonhomologous end-joining (NHEJ)
• Mutations in ATM gene on chromosome 11
• Ataxia Telangiectasia Mutated gene
• Repairs double stranded DNA breaks via NHEJ
• DNA hypersensitive to ionizing radiation
• CNS, skin, immune system affected
Ataxia Telangiectasia
Clinical Features

• Most children healthy for first year

• Begin walking at normal age but slow development
• Progressive motor coordination problems
• By 10 years old, most in wheelchairs
• Other symptoms
• Recurrent sinus/respiratory infections (immune system)
• Telangiectasias (skin)
• High risk of cancer
• DNA repair can also fail, due to inherited
mutations, there by leading to predisposition to
cancer. Examples are tumor supressor gene
inactivation either through mutation or deletion.
• P53 gene encodes a protein that prevents a cell
with damaged DNA from entering S-phase.
• It is associate with a disease called Li Fraumeni
syndrome.
LI FRAUMENI SYNDROME.
• Ataxia telangiectasia mutated (ATM) is
a serine/threonine protein kinase that is
recruited and activated by DNA double-strand
breaks. It phosphorylates several key proteins
that initiate activation of the DNA
damage checkpoint, leading to cell cycle
arrest, DNA repair or apoptosis.
 FANCONI ANAEMIA
• Fanconi anaemia (FA) is a rare genetic disease.
Among those affected the majority develop cancer,
most often acute myelogenous leukemia, and 90%
develop bone marrow failure (the inability to
produce blood cells) by age 40. About 60–75% of
people have congenital defects, commonly short
stature, abnormalities of the skin, arms, head, eyes,
kidneys, and ears, and developmental disabilities.
Around 75% of people have some form of endocrine
problem, with varying degrees of severity.
 Question:
• What would be the
complementary DNA
strand for the following
DNA sequence?

DNA 5’-CGTATG-3’

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 Answer:

DNA 5’-CGTATG-3’
DNA 3’-GCATAC-5’

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