Biosynthesis of Nucleic
Acids: Replication
Chapter 10
Flow of Genetic Information in the Cell
What is Replication of DNA?

Duplication of DNA – Replication

Giving rise to a new DNA molecule with
same base sequence as original

Mitosis – produces daughter cells
What is Replication of DNA?

Replication involves separation of the two
original strands and synthesis of two new
daughter strands using the original strands as
templates
Challenges of Replication of circular DNA




Continuous unwinding and separation of the
two DNA strands
Protection of unwound portions from attack
by Nucleases
Synthesis of the DNA template from one 5’ ->
3’ strand and one 3’ -> 5’ strand
Efficient protection from errors
What is Semi-Conservative Replication?

Each daughter strand
contains one template
strand and one newly
synthesized strand
DNA Replication- Matthew Meselson and
Franklin Stahl




Bacterial cells were grown in a heavy isotope
of nitrogen 15N
All the DNA incorporated 15N
Cells were switched to media containing
lighter 14N
DNA was extracted from the cells at various
time intervals
7
8
Matthew Meselson and Franklin Stahl

Observed that
15N-DNA has a
higher density
than 14N-DNA,
and the two can
be separated by
density-gradient
ultracentrifugati
on
DNA Replication



The DNA from different time points was
analyzed for ratio of 15N to 14N it contained
After 1 round of DNA replication, the DNA
consisted of a 14N-15N hybrid molecule
After 2 rounds of replication, the DNA
contained 2 types of molecules:


half the DNA was 14N-15N hybrid
half the DNA was composed of 14N
10
DNA Replication
DNA replication includes:

Initiation – replication begins at an origin of
replication

Elongation – new strands of DNA are
synthesized by DNA polymerase

Termination – replication is terminated
differently in prokaryotes and eukaryotes
11
In which direction does replication go?




DNA double helix unwinds at a specific point origin of replication
Polynucleotide chains are synthesized in both
directions from the origin of replication
DNA replication is bidirectional in most
organisms
Two replication forks - points at which new
polynucleotide chains are formed
Prokaryotic DNA Replication

Replication begins at one origin of replication and
proceeds in both directions around the chromosome
(Replisome).
13
In which direction does replication go?

In eukaryotic chromosome - there are
several origins of replication and two
replication forks at each origin
Properties of DNA Polymerases
Functions of various DNA polymerases

DNA-Pol I: repair and patching of DNA

DNA-Pol III: responsible for the polymerization
of the newly formed DNA strand

DNA-Pol II, IV, and V: proofreading and repair
enzymes
17
Requirements of DNA polymerase

DNA polymerase function has the following
requirements:



all four deoxyribonucleoside triphosphates:
dTTP, dATP, dGTP, and dCTP
Mg2+
Primer - a short strand of RNA to which the
growing polynucleotide chain is covalently
bonded in the early stages of replication
Supercoiling and Replication

DNA gyrase : catalyzes
reaction involving relaxed
circular DNA:



creates a nick in relaxed
circular DNA
a slight unwinding at the
point of the nick
introduces supercoiling
the nick is resealed
Unwinding of supercoiled DNA

Helicase, a helix-destabilizing protein, promotes
unwinding by binding at the replication fork

Single-stranded binding (SSB) protein
stabilizes single-stranded regions by binding
tightly to them
Prokaryotic DNA Replication
The enzymes for DNA replication are contained
within the replisome.
The replisome consists of


the primosome - composed of primase and
helicase
2 DNA polymerase III molecules
The replication fork moves in 1 direction,
synthesizing both strands simultaneously.
21
Primase Reaction

The Primase reaction

RNA serves as a primer in DNA replication

Primer activity first observed in-vivo.

Primase - catalyzes the copying of a short stretch
of the DNA template strand to produce RNA
primer sequence
Synthesis of new DNA strands
Synthesis and linking of new DNA strands



begun by DNA polymerase III
the newly formed DNA is linked to the 3’-OH of
the RNA primer
as the replication fork moves away, the RNA
primer is removed by DNA polymerase I
Semidiscontinuous model for DNA
replication
25
Semidiscontinuous model for DNA
replication

DNA polymerase synthesizes the strands



the leading strand is synthesized continuously in the
5’ - 3’ direction toward the replication fork
the lagging strand is synthesized
semidiscontinuously (Okazaki fragments) also in the
5’ - 3’ direction, but away from the replication fork
lagging strand fragments are joined by the enzyme
DNA ligase
Summary of DNA Replication in
Prokaryotes



DNA synthesis is bidirectional
DNA synthesis is in the 5’ -> 3’ direction
 the leading strand is formed continuously
 the lagging strand is formed as a series of Okazaki
fragments which are later joined
Five DNA polymerases have been found to exist in E. coli
 Pol I is involved in synthesis and repair
 Pol II, IV, and V are for repair under unique conditions
 Pol III is primarily responsible for new synthesis
Summary of DNA Replication in
Prokaryotes



Unwinding
 DNA gyrase introduces a swivel point in advance of the
replication fork
 a helicase binds at the replication fork and promotes
unwinding
 single-stranded binding (SSB) protein protects exposed
regions of single-stranded DNA
Primase catalyzes the synthesis of RNA primer
Synthesis
 catalyzed by Pol III
 primer removed by Pol I
 DNA ligase seals remaining nicks
Proofreading and Repair

DNA replication takes place only once each generation in
each cell

Errors in replication (mutations) occur spontaneously
only once in every 109 to 1010 base pairs

Proofreading - the removal of incorrect nucleotides
immediately after they are added to the growing DNA
during replication (Figure 10.10)

Errors in hydrogen bonding lead to errors in a growing
DNA chain once in every 104 to 105 base pairs
Proofreading Improves Replication
Fidelity

Cut-and-patch catalyzed by Pol I: cutting is
removal of the RNA primer and patching is
incorporation of the required deoxynucleotides

Nick translation: Pol I removes RNA primer or
DNA mistakes as it moves along the DNA and
then fills in behind it with its polymerase activity
DNA Polymerase Repair
Proofreading Improves Replication
Fidelity

Mismatch repair: enzymes recognize that two
bases are incorrectly paired, the area of mismatch
is removed, and the area replicated again

Base excision repair: a damaged base is
removed by DNA glycosylase leaving an AP site;
the sugar and phosphate are removed along with
several more bases, and then Pol I fills the gap
Mismatch Repair in Prokaryotes
Nucleotide-Excision Repair





Common for DNA lesions caused by UV
light or chemical means
Leads to deformed DNA
Removed by ABC excinuclease
Common repair for ultraviolet damage in
mammals
Xeroderma pigmentosum
Eukaryotic DNA Replication




More complicated
Multiple origins of
replication
Timing of cell
divisions must be
controlled
More proteins and
enzymes are involved
Eukaryotic DNA Replication

Multiple origins of replication – Replicators

Specific DNA sequences present between
gene sequences.

Zones where replication is proceeding Replicons
How is replication tied to cell
division?

Best understood model
for control of
eukaryotic replication
is from yeast.

DNA replication
initiated by
chromosomes that have
reached the G1 phase
How is replication tied to cell
division?

Replication initiated by Origin Recognition
Complex (ORC)

Activation factor – Replication Activator
Protein (RAP)

Replication Licensing factors (RLF) bind

Pre-Replication Complex (Pre-RC)
How is replication tied to cell
division?





Cyclins + Cyclin dependent protein kinases
Phosphorylate - activate DNA replication
and block assembly of Pre-RC
RAP and RLFs are degraded
G2 phase – DNA replication
M phase –DNA is separated into daughter
cells
Eukaryotic DNA Polymerases

At least 15 different polymerases are present in
eukaryotes (5 have been studied more extensively)
The Eukaryotic Replication Fork
Structure of the PCNA Homotrimer

PCNA is the eukaryotic equivalent of the part of Pol III
that functions as a sliding clamp ().



This project is funded by a grant awarded under the President’s Community Based Job Training Grant as implemented by
the U.S. Department of Labor’s Employment and Training Administration (CB-15-162-06-60). NCC is an equal
opportunity employer and does not discriminate on the following basis:
against any individual in the United States, on the basis of race, color, religion, sex, national origin, age disability,
political affiliation or belief; and
against any beneficiary of programs financially assisted under Title I of the Workforce Investment Act of 1998
(WIA), on the basis of the beneficiary’s citizenship/status as a lawfully admitted immigrant authorized to work in the
United States, or his or her participation in any WIA Title I-financially assisted program or activity.
Disclaimer

This workforce solution was funded by a grant awarded under the
President’s Community-Based Job Training Grants as implemented by the
U.S. Department of Labor’s Employment and Training
Administration. The solution was created by the grantee and does not
necessarily reflect the official position of the U.S. Department of
Labor. The Department of Labor makes no guarantees, warranties, or
assurances of any kind, express or implied, with respect to such
information, including any information on linked sites and including, but
not limited to, accuracy of the information or its completeness, timeliness,
usefulness, adequacy, continued availability, or ownership. This solution
is copyrighted by the institution that created it. Internal use by an
organization and/or personal use by an individual for non-commercial
purposes is permissible. All other uses require the prior authorization of
the copyright owner.