BBT312

Molecular Biology

Lecture #7
Watson & Others
Molecular Biology of the Gene
Chapter 9
DNA replication: Part I
 DNA Replication
DNA replication results in the synthesis of new DNA
DNA replication is the biological process of producing two identical replicas of DNA from
one original DNA molecule. This process occurs in all living organisms and is the basis for
biological inheritance.(https://en.wikipedia.org/wiki/DNA_replication)

DNA has an antiparallel

double helix structure, the
nucleotide bases are
hydrogen bonded together,
and each strand
complements the other. DNA
is replicated in a semi-
conservative manner, each
strand is used as the
template for the newly made
strand.

https://bio.libretexts.org/Courses/University_of_California_Davis/BIS_2A Q1. What is DNA replication? Why is it called

%3A_Introductory_Biology_-_Molecules_to_Cell/Spring_2020_BisA2_Facciotti/ a semi-conservative process?
 DNA Replication
DNA synthesis requires Deoxynucleoside Triphosphates and a Primer:Template junction

Q1. What are primers?

Q2. What is the importance of primer for DNA synthesis?
 DNA Replication
DNA synthesis requires Deoxynucleoside Triphosphates and a Primer:Template junction

Q1. What are the substrates required for the synthesis of DNA?
 DNA Replication
DNA is synthesized by extending the 3’-end of the primer

Q1. Explain how DNA is synthesized using a primer.

 DNA Replication
DNA is synthesized by extending the 3’-end of the primer
DNA polymerase catalyzes the addition of
the 5' phosphate group from an incoming
nucleotide to the 3' hydroxyl group of the
previous nucleotide. This process creates a
phosphodiester bond between the
nucleotides while hydrolyzing the
phosphoanhydride bond in the nucleotide.

In 1956, Arthur Kornberg purified the

first protein from E. coli that can
https://bio.libretexts.org/Courses/University_of_California_Davis/BIS_2A
synthesize DNA. This enzyme was %3A_Introductory_Biology_-_Molecules_to_Cell/Spring_2020_BisA2_Facciotti/
2020_Spring_Bis2a_Facciotti_Lecture_20

named as DNA polymerase I.

Q1. Diagrammatically show the mechanism of DNA synthesis.
(The diagram on the following slide can be used to answer this question)
 DNA Replication
Driving force for DNA synthesis is hydrolysis of Pyrophosphate (PPi)

Q1. Explain how pyrophosphate hydrolysis drives DNA synthesis.

 DNA Replication
DNA polymerases use a single active site to catalyze DNA synthesis
The synthesis of DNA is catalyzed by a class of enzymes called DNA polymerases. Unlike
most enzymes, which have one active site that catalyzes one reaction, DNA polymerase uses
a single active site to catalyze the addition of any of the four deoxynucleoside triphosphates.
The DNA polymerase monitors the ability of the incoming nucleotide to form an A:T or G:C
base pair, rather than detecting the exact nucleotide that enters the active site (Fig. 9-3).

Q1. What is required for DNA polymerase-

catalyzed nucleotide addition?
Q2. Diagrammatically show how correct base pairs
are incorporated by DNA polymerases in their
active sites.
 DNA Replication
Steric Constrains prevent DNA polymerase to incorporate ribonuclotides
DNA polymerases show an impressive ability to distinguish between ribonucleoside and
deoxyribonucleoside triphosphates (rNTPs and dNTPs).
This discrimination is mediated by the steric exclusion of rNTPs from the DNA polymerase
active site (Fig. 9-4). In DNA polymerase, the nucleotide-binding pocket cannot accommodate
a 2’-OH on the incoming nucleotide. This space is occupied by two amino acids that make
van der Waals contacts with the sugar ring.

Q1. Explain with a diagram how DNA polymerase prevents incorporation of rNTPs in a growing chain of DNA.
 DNA Replication
DNA polymerases resemble a hand that grips the primer:template junction

Q1. Diagrammatically show how DNA polymerase resembles a right hand.

No question in exam from Figure 9-5 (b).
 DNA Replication
DNA polymerases are Processive Enzymes

Q1. Why DNA polymerases are called processive enzymes?

Q2. What is the meaning of the degree or processivity of DNA polymerases?
Q3. Diagrammatically show how DNA polymerases synthesize DNA in a
processive manner.
 DNA Replication
Exonucleases proofread newly synthesized DNA

https://www.mun.ca/biology/scarr/Keto_vs_Enol.html

Q1. Discuss how tautomeric shift of guanine results in mispairing with thymidine.
 DNA Replication
Mechanism of action of Proofreading Exonucleases

Q1. Discuss the mechanism of proofreading

exonuclease activity of DNA polymerase.
Q2. What is the difference between exonuclease and
endonuclease?
 DNA Replication
Both strands of DNA are synthesized together at the replication fork
The junction between the newly separated template strands and the un-replicated duplex
DNA is known as the replication fork (Fig. 9-12).
The antiparallel nature of DNA creates a complication for the simultaneous replication of the two
exposed templates at the replication fork. Because DNA is synthesized only by elongating a 3’-end, only
one of the two exposed templates can be replicated continuously as the replication fork moves. On this
template strand, the polymerase simply “chases” the moving replication fork. The newly synthesized
DNA strand directed by this template is known as the leading strand.
Synthesis of the new DNA strand directed by the other ssDNA template is more complicated. This
template directs the DNA polymerase to move in the opposite direction of the replication fork. The new
DNA strand directed by this template is known as the lagging strand. As shown in Figure 9-12, this
strand of DNA must be synthesized in a discontinuous fashion.

Q1. Diagrammatically show the replication fork on

a replicating DNA.
Q2. What are leading strands and lagging strands?
Q3. What are Okazaki fragments?
 DNA Replication
Simultaneous DNA synthesis in both leading and lagging strands during DNA synthesis (Craig)
Since the DNA polymerase can only synthesize DNA in a 5' to 3' direction, the polymerization of
the strand opposite of the leading strand must occur in the opposite direction that helicase, or
front of the replication fork, is traveling. This strand is called the lagging strand, and due
to geometric constraints, must be synthesized through a series of RNA priming and DNA
synthesis events into short segments called Okazaki fragments.

Q1. Diagrammatically show

the replication fork on a
replicating DNA.
Q2. What are leading strands
and lagging strands? https://bio.libretexts.org/Courses/University_of_California_Davis/BIS_2A%3A_Introductory_Biology_-
_Molecules_to_Cell/Spring_2020_BisA2_Facciotti/2020_Spring_Bis2a_Facciotti_Lecture_208
Q3. What are Okazaki
fragments?
 DNA Replication
Simultaneous DNA synthesis in both leading and lagging strands during DNA synthesis (Klug/Craig)

https://www.mun.ca/biology/scarr/Leading_&_Lagging.html

No question in exam from this slide.

 DNA Replication
The Initiation of a new strand of DNA requires an RNA primer
All DNA polymerases require a primer with a free 3’-OH. They cannot initiate a new DNA
strand de novo. How, then, are new strands of DNA synthesis started?
To accomplish this, the cell takes advantage of the ability of RNA polymerases to do what
DNA polymerases cannot: start new RNA chains de novo. Primase is a specialized RNA
polymerase dedicated to making short RNA primers (5–10 nucleotides long) on an ssDNA
template. These primers are subsequently extended by DNA polymerase. Although DNA
polymerases incorporate only deoxyribonucleotides into DNA, they can initiate synthesis
using either an RNA primer or a DNA primer annealed to the DNA template.

Q1. What is primase?

Q2. Why primase is important for
DNA replication?
Q3. How different is the frequency
of primase function between
leading and lagging strands?
Q4. Which factors ensure that
primase is active only in
replication fork?
 DNA Replication
RNA primers must be removed to complete DNA replication

Removal of the RNA primer leaves a gap in the dsDNA that is an ideal
substrate for DNA polymerase—a primer:template junction (see Fig. 9-13).
DNA polymerase fills this gap until every nucleotide is base-paired, leaving a
DNA molecule that is complete except for a break in the phosphodiester
backbone between the 3’-OH and 5’-phosphate of the repaired strand. This
“nick” in the DNA can be repaired by an enzyme called DNA ligase. DNA
ligases use high-energy co-factors (such as ATP) to create a phosphodiester
bond between an adjacent 5’-phosphate and 3’-OH. Only after all RNA
primers are replaced by DNA and the associated nicks are sealed is DNA
synthesis complete.

Q1. Name the protein that is involved in the removal of RNA primers
from replicating DNA.
Q2. Name all the proteins involved in the removal of RNA primers
and completion of DNA replication in newly synthesized DNA.
Q3. Discuss the roles of DNA polymerase and DNA ligase to complete
DNA replication after removal of RNA primers by RNaseH.
 DNA Replication
DNA Helicases unwind the double helix in advance of the replication fork

Q1. What is the function of DNA helicases during DNA replication?

Q2. Diagrammatically show how DNA helicases separate two strands of the double helix.
 DNA Replication
Single stranded DNA (ssDNA) binding proteins (SSBs) stabilize ssDNA before replication

Q1. Discuss the roles of single-stranded DNA binding (SSB)

proteins during DNA replication. How do they interact with DNA?
 DNA Replication
Topoisomerases remove supercoils produced by DNA unwinding at the replication fork
As the strands of DNA are separated at the replication fork, the dsDNA in front of the fork becomes
increasingly positively supercoiled (Figure 9-17).

The supercoils introduced by the action of the DNA helicase are

removed by topoisomerases that act on the un-replicated dsDNA in
front of the replication fork (Fig. 9-17). These enzymes do this by
breaking either one or both strands of the DNA without letting
go of the DNA and passing the same number of DNA strands
through the break (as we discussed in Chapter 4). This
action relieves the accumulation of supercoils. In this way,
topoisomerases act as a “swivelase” that prevents the
accumulation of positive supercoils ahead of the replication fork.

This problem is most clear for the circular chromosomes of

bacteria, but it also applies to eukaryotic chromosomes. Because
eukaryotic chromosomes are not closed circles, they could in
principle rotate along their length to dissipate the introduced
supercoils. This is not the case, however: It is simply not possible
to rotate a DNA molecule that is millions of base pairs long each
time one turn of the helix is unwound.

FIGURE 9-17 Action of topoisomerase Q1. Diagrammatically show the action of

at the replication fork. topoisomerase at the replication fork.
 DNA Replication
Replication fork enzymes extend the range of DNA polymerase substrates

Q1. Name the proteins that function at the replication

fork of (i) E. coli and (ii) humans.
 Next Lecture:
DNA Replication: Part II

Reference Textbook:
Molecular Biology of the Gene
By Watson et al.
Chapter 9