DNA Replication and Repair

Learning Objectives
• Student will be able to

– Discuss DNA Replication

– Explain DNA Repair
– Solve clinical problem
 DNA Replication
• The process of making an identical new
DNA copy of a duplex (double-stranded)
DNA, using existing DNA as a template.

• In humans and other eukaryotes,

replication occurs in the cell nucleus.
 DNA Replication
• The genetic information found in DNA is
copied and transmitted to daughter cells
through DNA replication.
Do you have any Idea about
process of DNA Replication ?
The flow of
information from
DNA to RNA to
protein is termed
as “central
dogma” of
molecular biology
 Secret behind Central Dogma
• The Central Dogma. This states that once
‘information’ has passed into protein it cannot
get out again. In more detail, the transfer of
information from nucleic acid to nucleic acid, or
from nucleic acid to protein may be possible, but
transfer from protein to protein, or from protein to
nucleic acid is impossible. Information means
here the precise determination of sequence,
either of bases in the nucleic acid or of amino
acid residues in the protein.
 DNA Replication
• DNA replication takes
place by separation of
the strands of the
double helix, and
synthesis of two
daughter strands
complementary to the
two parental
templates.
 DNA Replication

– DNA replication is
called
semiconservative
because half of the
parent structure is
retained in each of
the daughter
duplexes.
Separation of the two DNA Strands
• For the initiation of Replication Process
• The two strands of the parental double
stranded DNA dsDNA must first separate
(or melt) over a small region.
• Because polymerase use only ssDNA as a
template.
 In prokaryotes
• DNA replication begins at a single unique
nucleotide sequence, a site called the
origin of the replication , or ori.
• The ori includes short , AT-rich segments
that facilitate melting.
 In eukaryotes
• Replication begins at multiple sites along
the DNA helix. Having multiple origins of
replication
• Which provides the mechanism for rapidly
replicating the great length of eukaryotic
DNA molecule.
 Proteins Required for DNA strand Separation

• Initiation of replication requires the

recognition of the origin by a group of the
protein that forms a prepriming complex.
• These proteins are
• DNA A protein
• DNA helicases
• Single stranded DNA –binding protein
 DNA A Protein
• This protein binds to specific nucleotide
sequences (DnaA boxes) within the origin
of replication, causing tandamly arranged
(one after the other) AT-rich regions in the
origin to melt.
• Melting is adenosine triphosphate (ATP)
dependent and results in strand separation
with the formation of localized region of
ssDNA.
 DNA helicases
• These enzymes bind to ssDNA near the
replication fork and then move into the
neighboring double stranded region,
forcing the strands apart (in effect ,
unwinding the double helix).
• Helicases require energy provided by
ATP.
• Unwinding at the replication fork causes
supercoiling in other regions of the DNA
molecule.
Single stranded DNA-binding protein
• This protein binds to the ssDNA generated
by helicases.
• The SSB protein are not enzymes, but
rather serve to shift the equilibrium
between dsDNA and ssDNA in the
direction of a single stranded forms.
• These proteins not only keep the strands
of DNA separated in the area of the
replication origin, but also protects the
Dna from nucleases that degrade ssDNA
 Supercoiling
• As the two strands of double helix are
separated a problem is encountered
namely, the appearance of positive
supercoils in the region of DNA ahead of
the replication fork as a result of over
winding.
• And negative supercoils in the region
behind the fork.
• The accumulating positive supercoils
interfere with further unwinding of the
double helix.
• To solve this problem there is a group of
enzymes called Dna topoisomerases
responsible for removing supercoils in the
helix by transiently cleaving one or both of
the DNA strand.
 Type I DNA topoisomerases
• These enzyme reversibly cleaves one
strand of the double helix.
• They have both strand cutting and strand
resealing activities.
• They do not require ATP but store energy
from phosphodiester bond they cleave.
• Reuse the energy to reseal the strand
Each time a transient
“nick” is
created in one DNA
strand, the intact DNA
strand is passed
through the break
before it is resealed,
thus relieving
(“relaxing”)
accumulated
supercoils.
 Type II DNA topoisomerases
• These enzymes bind tightly to the DNA double
helix and make transient breaks in both strands.
• The enzyme then causes a second stretch of the
DNA double helix to pass through the break and,
finally, reseals the break (Figure 29.13). As a
result, both negative and positive supercoils can
be relieved by this ATP-requiring process.
 Direction of DNA replication
• The DNA polymerases responsible for copying
the DNA templates are only able to “read” the
parental nucleotide sequences in the 3'→5'
direction, and they synthesize the new DNA
strands only in the 5'→3' (antiparallel) direction.
• Therefore, beginning with one parental double
helix, the two newly synthesized stretches of
nucleotide chains must grow in opposite
directions—
• one in the 5'→3' direction toward the replication
fork and one in the 5'→3‘ direction away from
the replication fork.
• Leading strand: The strand that is being copied
in the direction of the advancing replication fork
is called the leading strand and is synthesized
continuously.
• Lagging strand: The strand that is being copied
in the direction away from the replication fork is
synthesized discontinuously, with small
fragments of DNA being copied near the
replication fork.
• These short stretches of discontinuous DNA,
termed Okazaki fragments, are eventually joined
(ligated) to become a single, continuous strand.
The new strand of DNA produced by this
mechanism is termed the lagging strand
 RNA primer
• DNA polymerases cannot initiate synthesis
of a complementary strand of DNA on a
totally single-stranded template. Rather,
they require an RNA primer—that is, a
short, double-stranded region consisting of
RNA base-paired to the DNA template,
with a free hydroxyl group on the 3'-end of
the RNA strand (Figure 29.15).
• This hydroxyl group serves as the first
acceptor of a deoxynucleotide by action of
DNA polymerase.
 Primase
• A specific RNA polymerase, called primase
(DnaG), synthesizes the short stretches of RNA
(approximately ten nucleotides long) that are
complementary and antiparallel to the DNA
template. In the resulting hybrid duplex, the U in
RNA pairs with A in DNA. As shown in Figure,
these short RNA sequences are constantly
being synthesized at the replication fork on the
lagging strand, but only one RNA sequence at
the origin of replication is required on the leading
strand.
• The substrates for this process are 5'-
ribonucleoside triphosphates, and
pyrophosphate is released as each
ribonucleoside monophosphate is added
through formation of a 3'→5‘
phosphodiester bond.
• [Note:The RNA primer is later removed]
 Primosome
• The addition of primase converts the
prepriming complex of proteins required
for DNA strand separation to a
primosome. The primosome makes the
RNA primer required for leading strand
synthesis, and initiates Okazaki fragment
formation in lagging strand synthesis. As
with DNA synthesis, the direction of
synthesis of the primer is 5'→3'.
 Chain elongation
• Prokaryotic (and eukaryotic) DNA
polymerases elongate a new DNA strand
by adding deoxyribonucleotides, one at a
time, to the 3'- end of the growing chain.
• The sequence of nucleotides that are
added is dictated by the base sequence of
the template strand with which the
incoming nucleotides are paired.
 DNA polymerase III
• DNA chain elongation is catalyzed by DNA
• polymerase III. Using the 3'-hydroxyl group
of the RNA primer as the acceptor of the
first deoxyribonucleotide, DNA polymerase
III begins to add nucleotides along the
single-stranded template that specifies the
sequence of bases in the newly
synthesized chain.
• DNA polymerase III is a highly
processive” enzyme—that is, it remains
bound to the template strand as it moves
along, and does not diffuse away and then
rebind before adding each new nucleotide.
The processivity of DNA polymerase III is
the result of its β subunit forming a ring
that encircles and moves along the
• template strand of the DNA, thus serving
as a sliding DNA clamp.
• The new strand grows in the 5 – 3
direction , antiparallel to the parental
strand.
• All four substrates deoxyadenosine
triphosphate, deoxythymidine triphosphate
, deoxycytidine triphosphate and
deoxyguanosinetriphosphate must be
present for DNA elongation to occur. If any
one in short supply DNA synthesis will
stop.
 DNA Replication
• Exonuclease Activities of DNA
Polymerases
– DNA polymerase I is involved in DNA repair
and also removes RNA primers and replaces
them with DNA.
– Exonucleases degrade nucleic acids by
removing 5’ or 3’ terminal nucleotides.
The exonuclease activities of
DNA polymerase I
 DNA Replication (18)
• Initiation of Replication in Eukaryotic Cells
– Eukaryotes replicate their genome in small portions
(replicons).
– Initiation of DNA synthesis in a replicon is regulated.
 DNA Replication
• The Eukaryotic Replication Fork
– Replication activities are similar in eukaryotes
and prokaryotes.
– There are several DNA polymerases in
eukaryotes.
– Eukaryotic DNA polymerases elongate in the
5’-to-3’ direction and require a primer; some
have 3’-to-5’ exonuclease activity.
Some Proteins Required for Eukaryotic
DNA Replication
 DNA Repair
• DNA repair is essential for cell survival.
– DNA is the cell molecule most susceptible to
environmental damage.
– Ionizing radiation, common chemicals, UV
radiation and thermal energy create
spontaneous alteration (lesions) in DNA.
– Cells have a number of mechanisms to repair
genetic damage.
A pyrimidine dimer that has formed within a
DNA duplex following UV irradiation
– Nucleotide excision repair
(NER) removes bulky lesions,
such as pyrimidine dimers
and chemically altered
nucleotides.
– It consists of two pathways:
• A transcription-coupled
pathway which is the
preferential pathway and
selectively repairs genes
of greatest importance to
the cell.
• A global genomic pathway
which is less efficient and
corrects DNA strands in
the remainder of the
genome.
 DNA Repair
• Nucleotide excision repair (continued)
– TFIIH is a key component of the repair
machinery and is also involved in the initiation
for transcription. It links transcription and DNA
repair.
– A pair of endonucleases cut on both sides of
the lesion, and the damaged strand is
removed by helicase.
– The gap is filled by a DNA polymerase and
sealed by DNA ligase.
 DNA Repair
• Base Excision Repair
– Base excision repair (BER) removes altered
nucleotides that produce distortions of the
double helix.
– DNA glycosylase recognizes the alteration
and cleaves the base form the sugar.
– DNA glycosylases are specific for a particular
type of altered base.
Base excision repair
DNA glycosylase
removes the altered
bases.
Once the altered base
is removed, an
endonuclease cleaves
the DNA backbone and
a polymerase fills the
gap by inserting a
nucleotide
complementary to the
undamaged strand.
The strand is sealed by
DNA ligase
 DNA Repair
• Mismatch repair (MMR) is the correction
of mistakes that escape the DNA
polymerase proofreading activity.
– Repair enzymes recognize distortions caused
by mismatched bases.
– In bacteria, the parental strands are
recognized from daughter strands by the
presence of methylated bases.
– Several MMR pathways have been identified
in eukaryotes.
 DNA Repair
• Double-Strand Breakage Repair
– Ionizing radiation (X-rays, gamma rays) along
with some chemicals cause double-strand
breaks (DSBs).
– DSBs can be repaired by a pathway in
mammalian cells called nonhomologous end
joining (NHEJ) in which proteins bind to the
broken ends and catalyze reaction to rejoin
the broken ends.
Repairing DSBs by NHEJ
 DNA Repair
• Double-strand breakage repair (continued)
– Cells that lack one of the proteins required for
NHEJ are very sensitive to ionizing radiation.
– Another DSB repair pathway is homologous
recombination, and requires a homologous
chromosome to serve as a template for repair
of the broken strand.
– Defects in both repair pathways have been
linked to increased cancer susceptibility.
 The Human Perspective: The Consequences of
DNA Repair Deficiencies (1)

• Xeroderma pigmentosum (XP) patients

cannot repair sun-damaged DNA.
• Some help for XP patients may become
available in the form of skin creams that
contain DNA repair enzymes.
Xeroderma pigmentosum
The Human Perspective: The Consequences of
DNA Repair Deficiencies (2)

• Skin cells with optimal levels of repair

enzymes are subject to lesions that fail to
be excised and repaired.
• Skin cancer is not the only disease
promoted by deficiency or overworked
DNA repair systems.
• Some colon cancer cases are due to
mutations in mismatch repair genes.
 DNA Replication
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PRACTICE QUESTIONS
Q1. In replication, the template is read in
the direction of:-
a. 3’→5’
b. 5’→3’
c. 2’→3’
d. 3’→2’
Key: A
• The DNA template is read in 3′ to 5′ direction
whereas a new strand is synthesized in the 5′
to 3′ direction—this is often confused.
Q2. In a newly synthesized DNA strand,
the Primer is removed by:-
a. DNA polymerase I
b. DNA polymerase II
c. DNA polymerase III
d. DNA ligase
Key: A
a. DNA polymerase I: it removes the primer
b. DNA polymerase II: It catalyzes the transcription of
DNA to synthesize precursors of mRNA.
c. DNA polymerase III: it is the main polymerizing
enzyme in prokaryotes.
d. DNA ligase is an enzyme that repairs irregularities or
breaks in the backbone of double-
stranded DNA molecules
Q3.The section of DNA that codes for a
protein is:
a. Exon
b. Intron
c. Operon
d. Regulatory sequence
Key: A
a. Exon is a coding region.
b. Intron is a non-coding region
c. Operon is a functioning unit of DNA containing a
cluster of genes under the control of a single
promoter.
d. Regulatory sequence is a is capable of increasing or
decreasing the expression of specific genes
Q4 . The following enzyme unwinds the
DNA preceding the replication fork:-
a. Helicase
b. DNA Polymerase 1
c. Gyrase
d. Ligase
 Key: A
a. Helicase: unwinds the DNA
b. DNA Polymerase 1: it removes the primer
c. Gyrase: The enzyme causes negative
supercoiling of the DNA or relaxes positive
supercoils.
d. DNA ligase is an enzyme that repairs
irregularities or breaks in the backbone of
double-stranded DNA molecules
5. Which of the following type of bonds
lead to base pairing in DNA?

a. Hydrogen bonds between A=G

& T≡ C
b. Hydrogen bonds between A ≡T
& G=C
c. Covalent bonds between A = T &
G≡C
d. Hydrogen bonds between A=T
& G≡C
Key: D
• Adenine is always form bond with thymine
through two hydrogen bond and guinine
always form bond with cytosine with three
hydrogen bond.