DNA REPLICATION and REPAIR

By DR. PHYLIS C. RIO

 Learning Objectives
1. State importance of the study of replication
2. Trace the flow of genetic information
3. Define replication
4. Describe the process of DNA synthesis, giving
the roles of the enzymes involved
4.1. Explain and illustrate semiconservative
replication
4.2. Discuss the unwinding of the DNA helix
 4.3. Discuss the formation of the two kind
strands
5. Discuss the processes involved in the formation
of the new DNA chain giving roles of all the
enzymes involved
5.1. chain initiation
5.2. chain elongation
5.3. removal of RNA primer and ligation
6. Describe the different processes of DNA repair
 DNA Functions
• Replication and expression
• 1.DNA must be able to replicate itself so that
the information coded into its primary
structure is transmitted faithfully to progeny
• 2. The information must be expressed,
expression is through RNA intermediates
which in turn act as template for synthesis of
protein in body
 Central Dogma
• Shows relationship of DNA to RNA and to
protein
• Indicates three general mechanisms for
transfer of information
Replication ( DNA to DNA )
Transcription ( DNA to RNA )
Translation ( RNA to protein )
 DNA Replication
• Process by which two new strands of DNA are
made using the two existing strands as
templates
• Semiconservative – each strand of the
parental molecule serves as a template for
synthesis of a new strand; resulting daughter
DNA molecules consist of one intact template
strand ( half the original helix ) and a newly
replicated complementary strand
 Salient Features of Replication
• Each strand of DNA serves as a template over
which a new complementary strand is
synthesized
• Base pairing rule is always maintained. New
strand is joined to old strand by hydrogen bonds
between base pairs (A with T, G with C)
• Polymerization of the new strand of DNA is taking
place from 5’ to 3’ direction. This means that
template is read in 3’ to 5’ direction
• DNA polymerase synthesizes a new
complementary strand of DNA by
incorporating deoxynucleotide
monophosphate sequentially in 5’ to 3’
direction
• Both strands are replicated simultaneously
• Two double strands are produced in
replication. Each daughter cell gets only one
strand of the parent DNA. ( semiconservative)
 Steps Involved in DNA Replication in
Eukaryotes

1. Identification of the origins of replication

2. Unwinding ( denaturation ) of dsDNA to an
ssDNA template
3. Formation of the replication fork
4. Initiation of DNA synthesis and elongation
5. Formation of replication bubbles with ligation
of the newly synthesized DNA segment
6. Reconstitution of chromatin structure
 Classes of Proteins Involved in
Replication
PROTEIN FUNCTION
DNA polymerase Deoxynucleotide polymerization
Helicases Processive unwinding of DNA
Topoisomerases Relieve torsional strain that results from
helicase induce unwinding
RNA primase Initiates synthesis of RNA primer
Single-strand binding proteins Prevent premature reannealing of dsDNA
DNA ligase Seals the single strand nick between the
nascent chain and Okazaki fragments on
lagging strand
 Origin of Replication
• Specific site where replication begins ( Ori C)
• With series of direct repeat DNA sequence, rich
in A-T sequence, that bind to specific protein
( Dna A )
• Site at which initial separation of parental strands
occurs
• 150 -250 base pairs of DNA forms complex with
multimers of DNA- binding protein (DnaA )
• This leads to local denaturation and unwinding of
an adjacent A-T rich region of DNA
• In bacterial chromosome, origin of replication
is single and circular, replication process is
completed in 30 minutes
• In eukaryotes, origin of replication is multiple
(bubbles formation ) and linear, this is to
complete replication in a reasonable period of
time (approx. In 9 hours)
 DNA Unwinding
• Unwinding of DNA, provides a short region of
ssDNAs to which SSB and helicase binds; this
ssDNAs are essential for synthesis of nascent
DNA strands
• Single stranded DNA binding (SSB)protein
- is not an enzyme, also called helix
destabilizing protein
- binds only to single stranded DNA,
maintains seperation, protect DNA from
nucleases
• DNA Helicase
- catalyzes processive unwinding of double
helix of DNA
- requires energy from ATP
 Formation of Replication Fork
• Formed by a number of protein-protein and
protein-DNA interactions
• Four components of replication that form in
following sequence:
1.DNA helicase unwinds a short segment of
parental duplex DNA
2.A primase initiates synthesis of an RNA
molecule that is essential for priming DNA
synthesis
• 3. The DNA polymerase initiates nascent
daughter strand synthesis
• 4. SSBs bind to ssDNA and prevent premature
reannealing of ssDNA to dsDNA
 DNA Synthesis
• Template refers to structural sequence of the
polymerized monomeric units of a
macromolecules that provides the pattern for the
synthesis of another macromolecule with a
complementary or characteristic sequence
• Primer refers to a polymer molecule that
contains the growing point for further addition of
monomeric units
• In prokayrotes and eukaryotes, replication occurs
in both strands ( bidirection )
• DNA Polymerase III –functions at replication
fork, catalyzes the highest rate of chain
elongation and is the most processive
• DNA Polymerase II – mostly involved in
proofreading and DNA repair
• DNA Polymerase I – completes chain synthesis
between Okazaki fragments on lagging strand
 Comparison of Prokaryotic and
Eukaryotic DNA Polymerases
E. Coli Mammalian Function
I alpha Gap filling and synthesis of lagging strand
II epsilon DNA proofreading and repair
beta DNA repair
gamma Mitochondrail DNA synthesis
III delta Processive leading strand synthesis
 DNA Chain Initiation
• Initiation of synthesis requires priming by short
length of RNA (10 – 200 nucleotides )
• DNA polymerase cannot initiate synthesis of new
strands, requires primer
• Primer – an RNA oligonucleotide, synthesized by
RNA polymerase ( PRIMASE) in a 5’ to 3’
direction, copies DNA template strands
• RNA primers – are complementary to the
sequence on the strand of DNA template and
base pair with that portion of DNA
• DNA polymerase initially adds
deoxyribonucleotide to 3’ hydroxyl group of
primer
• Process involves nucleophilic attack by the 3’
hydroxyl group of the RNA primer on the
alpha phosphate of the first entering
deoxynucleoside triphosphate, with splitting
off of pyrophosphate
 DNA Chain Elongation

• DNA polymerase elongates new DNA strand by

adding deoxyribonucleotide, one at a time to the
3’ end of growing chain
• Nucleotide building blocks are 5’
deoxyribonucleotide triphosphate
• Incoming nucleotide added forms base pair with
complementary nucleotide on template strand,
an ester bond is formed with free 3’ hydroxyl
group at end of growing chain and
pyrophosphate is released
• Release of pyrophosphate and its subsequent
cleavage provide the energy for the
polymerization process
• Sequence is dictated by the base sequence of
template strand
• All four deoxyribonucleoside triphosphates
must be present for DNA elongation to occur
 Direction of DNA Synthesis
• DNA polymerase reads parental nucleotide
sequences in 3’ to 5’ direction and synthesizes
new DNA strands in 5’ to 3’ direction
• Leading strand is strand that grows in 5’ to 3’
direction, towards replication fork, synthesized
continuously
• Lagging strand is copied in 5’ to 3‘ direction away
from replication fork, synthesized discontinuously
with small fragment of DNA ( Okazaki fragment )
 Proofreading
• Misplaced nucleotide are hydrolytically
removed and replaced with correct nucleotide
• Excision is done opposite of synthesis DNA
( by3’ to 5’ exonuclease activity of
polymerase )
 Prevention of Supercoiling
1. Type I DNA Topoisomeras
- reversibly cuts a single strand of the double helix
- possesses both nuclease ( strand cutting ) and
ligase ( strand resealing ) activities
- makes transient “ nick “ which allows DNA helix to
rotate at phosphodiester bond opposite the nick
- do not requires ATP
2. Type II DNA Topoisomerase
- make transient breaks in both DNA strands
- causes a second stretch of DNA double helix to
pass through the break and reseals
 Termination of DNA Synthesis
• DNA polymerase III stops synthesis when it is
blocked by proximity to an RNA primer
• RNA is then excised and gap is filled by DNA
polymerase I
• As synthesis is being made, proofreading is also
being done to new chain using 3’ to 5’
exonuclease activity
• This removal, synthesis and proofreading
continues one nucleotide at a time, until RNA is
totally degraded and gap is filled with DNA
• DNA ligase catalyzes formation of
phosphodiester linkage between 5’ phosphate
group on DNA chain synthesized by DNA
polymerase III and 3’ hydroxyl group on chain
made by DNA polymerase I
• This process requires ATP
Reconstitution of Chromatin Structure
• Newly replicated DNA is rapidly assembled
into nucleosomes
 Summary of DNA Replication
• 1. Unwinding of parental DNA to form a
replication fork
• 2. RNA primer complimentary to DNA
template is synthesized by RNA primase
• 3. DNA synthesis is continuous in the leading
strand ( towards replication fork ) by DNA
polymerase
• 4. DNA synthesis is discontinuous in lagging
strand ( away from fork ) as Okazaki fragments
• 5. In both strands, synthesis is in 5’ to 3’
direction
• 6. When polymerization is complete, the RNA
primer are removed, gaps filled by
deoxynucleotide and strands are ligated by
DNA ligase
• 7. Proof reading is done by DNA polymerae
• 8. Finally organized into chromatin
DNA Synthesis Occurs During S Phase
• Replication occurs during S phase or synthetic
period
• Cell regulates its DNA synthesis grossly by
allowing it to occur only at specific times and
mostly in cells preparing to divide by a mitotic
process
• During S phase, DNA polymerase increases in
quantities
• Enzymes responsible for formation of substrates
for DNA synthesis ( ie, deoxyribonucleoside
triphosphate) are also increased in activity and
then decrease following S phase until the
reappearance of signal for renewed DNA
synthesis.
• During S phase, nuclear DNA is completely
replicated once and only once, it marked to
prevent its further replication until it again passes
through mitosis
 Cyclins
• Gene products that govern the transition from
one phase of the cell cycle to another
• These are a family of proteins whose
concentration increases and decreases
throughout the cell cycle
• These are turned on appropriate time, when
turned on , different cyclin dependent protein
kinases ( CDKs ) phosphorylate substrate
essential for progression through the cell cycle
Cyclin and Cyclin –dependent Kinases
Involved in Cell Cycle Progression
Cyclin Kinase Function
D CDK4, CDK6 Progression past restriction point at
G1/S boundary
E, A CDK2 Initiation of DNA synthesis in early S
phase
B CDK1 Transition from G2 to M
 Repair Mechanisms
• Used in persistent replication errors
• Used in damages by mutagens from
environment, physical and chemical agents

Mutagens – agents that DNA damage causing

mutation or cancer
 Types of Damage to DNA
• I. Single base alteration
A. Depurination
B. Deamination of cytosine to uracil
C. Deamination of adenine to hypoxanthine
D. Alkylation of base
E. Insertion or deletion of nucleotide
F. Base-analog incorporation
• II. Two base alteration
A. UV light induced thymine (pyrimidine )
dimer
B. bifunctional alkylating agent cross
linkage
• III. Chain break
A. ionizing radiation
B. radioactive disintegration of backbone
element
C. oxidative free radical formation
• IV. Cross linkage
A. between bases in same or opposite
strands
B. between DNA and protein molecules (eg
histone)
 Mechanism of DNA Repair
Mechanism Problem Solution
Mismatch repair Copying errors ( single base or Methyl directed strand
two to five base unpaired loops) cutting, exonuclease
digestion and replacement
Base excision Spontaneous, chemical or Base removal by N
repair radiation damage to a single base glycosylase, abasic sugar
removal, replacement
Nucleotide Spontaneous, chemical or Removal of an approximately
excision repair radiation damage to a DNA 30 nucleotide oligomer and
segment replacement
Double strand Ionizing radiation, chemotherapy, Synapsis, unwinding,
break repair oxidative free radicals alignment, ligation
 Basic Steps DNA Repair
• 1. Recognition of distortion / damage in DNA
• 2. Removal of portion / region with distortion
• 3. Filling up of gap by DNA polymerase using
undamaged strand as template
• 4. Sealing of nick by ligase
 Mismatch Repair
• Repair error made when DNA is copied
• Bases are not paired correctly, or 2 to 5 extra unpaired
bases are inserted due to polymerase slip or stutter
• Steps in repair:
- In bacteria, a specific protein scans using adenine
methylation within GATC sequence as reference point
- Template strand is methylated and newly
synthesized is not
- Difference allows repair enzymes to identify strand
with errant nucleotide that requires replacement
 - GATC endonuclease cuts strand with the
mutation
- Faulty DNA is removed by an exonuclease
- Defect is filled according to the pairing rule and
then ligated
In mammalian or human, error can also be
distinguish and then repair mismatch but the
process more complicated
Hereditary Nonpolyposis Colon Cancer ( HNPCC )-
linked to faulty mismatch repair mechanism
 Base Excision Repair
• Repairs damage to single base ( depurination /
depyrimidination) or occurrence of not normal
base
• Depurination of DNA is due to thermal lability of
purine N glycosidic bond
• Bases may be deaminated to form abnormal
bases, cytosine – uracil, adenine – hypoxanthine,
guanine – xanthine
Steps in repair:
- Abnormal base is recognized and removed by N
glycosylase
- Removal marks site of defect and allows an
apurinic or apyrimidinic endonuclease to
excise the abasic sugar
- Proper base is then replaced by DNA
polymerase and a ligase returns DNA original
base
 Nucleotide Excision Repair
• Mechanism is used to repair and replace
region of damaged DNA up to 30 bases in
length
• Repair damaged caused by the following:
1.UV light which induces formation of
cyclobutane pyrimidine-pyrimidine dimers
2.Smoking which causes formation of benzo[a]
pyreo-guanine adducts
3. Other causes – ionizing radiation, cancer
chemotherapeutic agents and variety of
chemicals that cause base modification, strand
breaks, cross linkage between bases on opposite
strands or between DNA and protein
Example best studied are pyrimidine dimers
formation caused by UV damage, adjacent
pyrimidine residues in DNA become covalently
linked ( Xeroderma pigmentosa )
• Steps in repair
1.Defect is detected and unwinding of strand is
made
2.Hydrolysis of 2 phosphodiester bonds in
strand defect by excision nuclease
(exinuclease )
3.Fragment of DNA 27 to 20 nucleotides long is
excised
• 4. strand is removed and replaced by base
pairing by polymerase and then joined to
existing strand by DNA ligase
 Double Strand Break Repair
• Mechanism is part of physiologic process of
immunoglobulin gene rearrangement
• It is also used to repair damage caused by
ionizing radiation and oxidative free radical
generation
• Involves 2 proteins; Ku- with latent ATP
dependent helicase and DNA – PK ( DNA
Protein Kinase )
• Steps in repair
1. Ku binds to free DNA ends
2. DNA-PK is recruited by KU, bind to free DNA
ends
3. There is approximation of two separate
ends
4. DNA –PK reciprocally phosphorylate Ku and
other DNA – Ku on opposite strand
• 5. DNA- PK dissociates from DNA – Ku,
resulting in activation of Ku helicase
• 6. Two end unwound
• 7. Unwound, approximated DNA form base
pair
• 8. Extra nucleotide tails are removed by
exonuclease and gaps filled and closed by DNA
ligase
 Inhibitors of DNA Replication
Inhibitors of DNA replication are valuable in the treatment of various types
of diseases
Used in antibiotics and anti cancer drugs
A. Inhibitors of Nucleotide Biosynthesis
- they interfere with the production of dTTP
- examples are methotrexate and fluorodeoxyuridylate

B. Inhibitors that Interact with DNA Template

- planar structure of drug binds noncovalently
between stacked of base pairs of duplex DNA, process is
called intercalation
- DNA function s poor template, thus affect fidelity of replication
- examples are actinomycin, acridine, ethidium
C. Nucleoside Analog Inhibitors
- block further chain growth at replication fork
- deoxynucleosides monophosphates are converted to triphosphates
- these are incorporated into 3’ hydroxyl end of g rowing DNA chain and
because the new end lacks 3’ hydroxyl, no further addition can occur

D. Inhibitors that Bind to Replication Proteins

- Aryldrazinopyrimidines are potent inhibitors of DNA polymerase III of
Gram (+) bacteria, form ternary complex with polymerase and DNA
template
- Aphidicolin, a tetracyclic diterpenoid is a potent inhibitor of mammalian
nuclear DNA polymerase
- Nalidixic acid and novobiocin bind to DNA gyrase to inhibit its action