DNA Replication

DNA replication occurs in all organisms and allows for genetic

inheritance. It can occur in a short period, copying up to approximately
ten to the 11th power (1011) units of information in some cases.
Semiconservative
• One parent or original strand of DNA is
distributed to each daughter duplex in
combination with a newly synthesized strand
with an antiparallel orientation. At the end of
the process, each of the two daughter strands
has half new DNA and half old DNA; thus, the
process is semiconservative (Figure DNA
synthesis is semiconservative with respect to
the parental strand).
Bidirectional with multiple origins of
replication
• This hastens the process of the replication
because replication begins at several sites on
linear DNA and is completed by the end of S
phase of the cell cycle. As replication nears
completion, “bubbles” of newly replicated
DNA come together forming two new
molecules (Figure Bidirectional with multiple
origins of replication).
Primed by short stretches of RNA
• A specific DNA polymerase–associated
enzyme, called DNA primase, synthesizes
short stretches of RNA that are
complementary and antiparallel to the DNA
template (fig Primed by short stretches of
RNA.)
Semidiscontinuous with respect to the
synthesis of new DNA
• All DNA polymerases “read” a parental strand 3′
to 5′ and synthesize a complementary
antiparallel new strand 5′ to 3′.
• The DNA polymerase synthesizes one strand in
the 5′ to 3′ continuously. This strand is called
the leading strand. The synthesis proceeds in
the same direction as replication fork
movement. The other new strand is synthesized
5′ to 3′, but discontinuously, creating fragments
that ligate (join) together later.
Semidiscontinuous with respect to the
synthesis of new DNA
• This strand is called the lagging strand and is
the one in which 5′ to 3′ synthesis proceeds
in the direction opposite to the direction of
the fork movement. The DNA synthesized on
the lagging strand as short fragments (100
to 200 nucleotides) is called the Okazaki
fragments. (Figure Semidiscontinuous with
respect to the synthesis of new DNA)
The Process of DNA
Replication
Initiation
• The origin of replication is a sequence of base
pairs in the genome where DNA replication
begins; these sequences tend to be high in AT
content making for easier separation. AT bonds
have the fewest hydrogen bonds, making them
weaker. Eukaryotes have multiple origins of
replication. Replication begins when origin-
binding proteins bind to the origin of replication
on the DNA.
Initiation
• Then the unwinding of the double helix proceeds by way of the
enzyme, helicase, creating a replication fork. The replication fork is Y-
shaped and is where the leading and lagging strands are formed.
Helicase begins unwinding by breaking hydrogen bonds. Single-
stranded binding proteins (SSBs) stabilize the unwound DNA,
preventing it from forming into secondary structures; the secondary
structures can prevent the continuation of the DNA polymerase.
Elongation
• Meanwhile, topoisomerases continue to open the DNA downstream
to allow for elongation. Once the DNA strand is open, there needs to
be a base for the DNA polymerase to bind and begin replication; this
is provided by the primase.
Elongation
• Primase adds short strands of RNA primers (9 to 12 pairs) onto the
template to allow for DNA polymerase alpha to bind and add
nucleotides. DNA polymerase alpha (in eukaryotes) is a complex that
has the DNA primase which creates the RNA primer, and then the
polymerase alpha itself elongates around 20 nucleotides and passes
off to DNA polymerase epsilon or delta.
Elongation
• DNA polymerase epsilon
elongates and proofreads
on the leading strand. DNA
polymerase delta elongates
and proofreads on the
lagging strand.
Elongation
• A new primer is added as the replication fork is further opened and the
DNA polymerase delta builds the new DNA strand away from the
replication fork, creating Okazaki fragments. Afterwards, another DNA
polymerase replaces the RNA primers with DNA. DNA ligase connects the
fragments.
Termination
• In eukaryotes replication ends at telomere regions. Telomeres are
regions at the end of chromosomes with repetitive nucleotides such
as TTAGGG sequences. Shortening telomeres have been associated
with cell aging and death.
DNA Proofreading
• The DNA replication is not perfect and has
mechanisms to ensure there are
corrections. DNA polymerases make
mistakes in 1 in 10^5 base pairs.
Considering the length of the human
genome is around 3 x 10^9 that is about
30,000 to 50,000 mistakes. However, with
the exonuclease activity of the
polymerases, the error rates are reduced
to a few hundred. These errors are further
corrected in the G2 phase of the cell cycle,
reducing the total number of errors to less
than 10.
PROTEINS INVOLVED IN DNA
SYNTHESIS
DNA polymerases
• several DNA polymerases are involved in
DNA replication and each of them
possesses distinct activities. They function
as a complex to initiate DNA synthesis.
Some DNA polymerases have 3′-5′
exonuclease activity, or proofreading ability,
that allows them to remove nucleotides
that are not part of the double helix. The
enzyme removes mismatched residues,
thus performing an editing function.
DNA Helicases
• DNA Helicases DNA helicases are
a class of motor proteins required
to unwind short segments of the
parental duplex DNA. These
enzymes catalyze the strand
separation and formation of the
replication fork during DNA
synthesis.
DNA Primases
• DNA primases initiate the
synthesis of an RNA molecule
essential for priming DNA
synthesis on both the leading and
the lagging strands. The first few
nucleotides are ribonucleotides
and the subsequent ones may be
either ribonucleotides or
deoxyribonucleotides.
Single-stranded DNA binding proteins
• Single-stranded DNA binding
proteins prevent premature
annealing of the single-stranded
DNA to double-stranded DNA.
DNA ligase
• DNA ligase is an enzyme that
catalyzes the sealing of nicks
(breaks) remaining in the DNA
after DNA polymerase fills the
gaps left by RNA primers. DNA
ligase is required to create the f
nal phosphodiester bond
between the adjacent nucleotides
on a strand of DNA.
Topoisomerases
• Topoisomerases Most cellular DNAs
have fewer right-hand turns than
expected from the number of their
base pairs. This underwound
condition (negative supercoils)
facilitates the unwinding of the
double helix during replication and
transcription. As the replication fork
moves along the helix, rotation of
the daughter molecules around one
another causes the DNA strands to
become overwound.
Topoisomerases
• The supertwisting of DNA can be
removed by enzymes known
collectively as topoisomerases.
These enzymes relieve torsional
stress in DNA by inducing
reversible single-stranded breaks
in DNA. The phosphodiester bond
either in one strand or in both
strands is cleaved initially. After
rotation of the DNA around its
axis, the enzyme seals the nick
Telomerase
• Telomerase is an enzyme that helps
to maintain the telomere. The
telomere, a protective repetitive
stretch of DNA complexed with
protein at the end of a chromosome,
shortens with every cell division.
Telomere shortening is recognized
as and is a part of the normal aging
process. Telomeres are important
chromosomal structures and allow
the cell to distinguish intact
chromosomes from broken
chromosomes and to protect
chromosomes from degradation.
Telomerase
• They also serve as substrates for
normal replication mechanisms.
In most organisms, telomeric DNA
consists of a tandem array of very
simple sequence of DNA (in
humans, it is TTAGGG).
• The telomere maintenance
enzyme, telomerase, is an RNA-
dependent DNA polymerase,
which adds TTAGGG repeats to
the ends of the chromosomes.
During eukaryotic DNA replication, topoisomerases
A. Catalyze the synthesis of an RNA primer on the lagging strand
B. Remove the incorrectly base-paired nucleotides through a 3′-5′
exonuclease activity
C. Stabilize single-stranded DNA in the region of the replication fork
D. Cut and reseal DNA in advance of the replication fork to eliminate
supercoiling
E. Add nucleotides to the growing strand in the 5′-3′ direction
• Correct answer = D. Topoisomerases remove torsion from replicating
DNA by cutting the double-stranded DNA, relaxing the supercoiling
followed by ligation. DNA primase catalyzes the addition of an RNA
primer during DNA synthesis. Proofreading activity is a property of
some DNA polymerases. Single-stranded DNA binding proteins
protect the DNA during replication by binding to the open template
strand. DNA polymerase synthesizes DNA in the 5′-3′ direction by
adding nucleotides to the growing chain.
Which of the following functions is associated with eukaryotic DNA
polymerase during DNA replication?
A. Continuous 5′-3′ DNA synthesis on the lagging strand
B. Energy-dependent formation of the replication fork
C. Proofreading newly synthesized DNA
D. Discontinuous 5′-3′ DNA synthesis on the leading strand
E. Removal of primers using 5′-3′ exonuclease activity
• Correct answer = C. Some of the DNA polymerases possess
proofreading ability, which is a 3′-5′ exonuclease activity. Continuous
DNA synthesis occurs on the leading strand and discontinuous
synthesis on the lagging strand. Helicase is necessary for breaking the
hydrogen bonds between the strands of DNA using ATP hydrolysis.
None of the DNA polymerases possess 5′-3′ exonuclease activity.
What is DNA replication?
a) Conservative
b) Non-conservative
c) Semi-conservative
d) None of the mentioned
• Answer: c
Explanation: Each DNA strand serves as a template for the synthesis
of a new strand, producing two new DNA molecules, each with one
new strand and one old strand.
What is the reaction in DNA replication catalyzed by DNA ligase?
a) Addition of new nucleotides to the leading strand
b) Addition of new nucleotide to the lagging strand
c) Formation of a phosphodiester bond between the 3’-OH of one
Okazaki fragment and the 5’-phosphate of the next on the lagging
strand
d) Base pairing of the template and the newly formed DNA strand
• Answer: c
Explanation: DNA ligase catalyzes the formation of a phosphodiester
bond between 3’-OH of one Okazaki fragment and 5’-phosphate of
the next.
Which of the following enzymes remove supercoiling in replicating
DNA ahead of the replication fork?
a) DNA polymerases
b) Helicases
c) Primases
d) Topoisomerases
• Answer: d
Explanation: Strand separation creates topological stress in the helical
DNA structure which is relieved by the action of topoisomerases.