The Central Dogma of a Cell

Dr S.Balakrishnan, PhD.,
Biochemistry 2/e - Garrett & Grisham

The Central Dogma of a Cell.

replication

Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham

The Central Dogma

DNA replicated into DNA transcribed into mRNA
translated into protein

Copyright © 1999 by Harcourt Brace & Company

 The Central Dogma of a Cell

• Genetic information is
transferred from DNA and
converted to protein

• RNA molecules work as

messengers

• Proteins are the biological

workers
• Information of the DNA is copied to a RNA
molecule in transcription

• RNA directs the

protein synthesis in a
translation

• Protein’s 3D structure
determines it’s
function

• Information transfer
only in one direction
 ORGANIZATION OF DNA:
(DNA Packaging in Eukaryotic Cell)

Dr Balakrishnan S, PhD.,
Dr Balakrishnan S, PhD.,
Dr Balakrishnan S, PhD.,
Dr Balakrishnan S, PhD.,
Dr Balakrishnan S, PhD.,
Nucleosomes and Chromatin:

Dr Balakrishnan S, PhD.,
 Denaturation and
Renaturation of DNA:
• Heat (>94oC), alkaline pH (>11.3), and
chemicals such as formamide and urea
are commonly used to denature DNA
• Disrupt hydrogen Bonding
• No covalent bonds (Phosphodiester
bonds) are broken
• Renatured (annealed) if the denaturing
condition is slowly removed

Dr Balakrishnan S, PhD.,
DNA Replication

Dr S.Balakrishnan, PhD.,
 DNA Replication
DNA replication is the process by which DNA
makes a copy of itself during cell division

14
 Models of DNA Replication
Matthew Meselson & Franklin Stahl, 1958
investigated the process of DNA replication
considered 3 possible mechanisms:
–conservative model
–semiconservative model
–dispersive model

15
Three possible models were proposed for DNA replication:
a. Conservative model proposed both strands of one copy would be
entirely old DNA, while the other copy would have both strands
of new DNA.
b. Dispersive model was that dsDNA might fragment, replicate
dsDNA, and then reassemble, creating a mosaic of old and new
dsDNA regions in each new chromosome.
c. Semiconservative model is that DNA strands separate, and a
complementary strand is synthesized for each, so that sibling
chromatids have one old and one new strand. This model was the
winner in the Meselson and Stahl experiment.
Fig. 3.1 Three models for the replication of DNA

台大農藝系 遺傳學 601 20000

Chapter 3 slide 17
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
 SALIENT FEATURES OF DNA REPLICATION

• Provision of progeny with the genetic make up possessed by the parents - primary
function.
• DNA replication must be complete, to be carried out in such a way as to maintain
genetic stability within the organism and the species.
• To ensure fidelity in replications, it involves many cellular functions. Several
proofreading procedures, and requires the formation of a number of protein-protein
and protein-DNA interactions.
• In all cells, replication can occur only from a single stranded DNA (ssDNA) template,
which occurs after unwinding double-stranded DNA (dsDNA).
• Mechanisms must exist to target the site of initiation of replication and to unwind the
double-stranded DNA (dsDNA) in that region, where the replication complex is
formed.
• After replication is complete in an area, the parent and daughter strand must reform
dsDNA.
• In Eukaryotic cells, an additional step is must, in that domain structure must reform,
including nucleosomes, that existed prior to the onset of replication.
Features of DNA Replication:
• DNA replication is bidirectional
– Bidirectional replication involves two replication forks, which move
in opposite directions
• DNA replication is semidiscontinuous
– The leading strand copies continuously
– The lagging strand copies in segments (Okazaki fragments) which
must be joined
An illustration to show replication of the leading and lagging strands of DNA.
 Enzymes involved in DNA Replication
 DNA helicase is the enzyme that unwinds the DNA double helix.
 DNA gyrase (topoisomerase II) helps unwind the DNA double helix
and keep the double strands from tangling during replication (release
the Torsional stress)
 RNA primase is the enzyme that builds an RNA primer on the parent
strand to initiate DNA replication
 DNA polymerases (Pol I, II, & III/ Pol α, β, γ, δ & ε) are the enzymes
that matches and lays down nucleotides to build the daughter DNA
strand along each parent DNA strand
 DNA ligase joins the adjacent Okazaki fragments on the lagging strand
of DNA
 Telomerase involves in synthesis of telomeres of eukaryotic DNA
Biochemistry 2/e - Garrett & Grisham
DNA Polymerase
Nucleotide polymerizing enzyme, first discovered in 1957

Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham

A comparison of prokaryotic and eukaryotic DNA polymerases.

Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham
Protein machinery for DNA replication

Copyright © 1999 by Harcourt Brace & Company

Steps and Proteins Involved in DNA Replication
 DNA Replication
DNA replication includes:
1. Initiation
2. Elongation
3. Termination

1. Initiation – replication begins at an origin of

replication
Ori binding protein (e.g. DnaA protein in E coli) binds to
repeats in ori, initiating strand separation at A+T rich
region, and DnaB, a helicase delivered by DnaC, further
unwinds. Primase then binds and constructs the RNA
primer.
2. Elongation – new strands of DNA are synthesized by
DNA polymerase
Elongation involves DnaB helicase unwinding, SSB binding to
keep strands separated, and DNA polymerase grinding away
on both strands

3. Termination – replication is terminated differently in

prokaryotes and eukaryotes
The "ter" locus, rich in Gs and Ts, signals the end of replication.
A Ter protein is also involved. Ter protein is a contrahelicase
and prevents unwinding
 Steps of DNA Replication:
1.The first step in DNA replication is to ‘unzip’ the double helix
structure of the DNA molecule.
2.This is carried out by an enzyme called helicase which breaks the
hydrogen bonds holding the complementary bases of DNA
together (A with T, C with G).
3.The separation of the two single strands of DNA creates a ‘Y’
shape called a replication ‘fork’. The two separated strands will
act as templates for making the new strands of DNA.
Supercoiling must be compensated - by DNA gyrase
4.One of the strands is oriented in the 3’ to 5’ direction (towards the
replication fork), this is the leading strand. The other strand is
oriented in the 5’ to 3’ direction (away from the replication fork),
this is the lagging strand.
As a result of their different orientations, the two strands are
replicated differently (shown in the following slides).
DNA replication Fork
 Leading Strand: Lagging strand:

5. A short piece of RNA called a 5. Numerous RNA primers are made

primer (produced by an enzyme by the primase enzyme and bind at
called primase) comes along and various points along the lagging
binds to the end of the leading strand.
strand. The primer acts as the
starting point for DNA synthesis. 6. Chunks of DNA, called Okazaki
6. DNA polymerase binds to the fragments, are then added to the
leading strand and then ‘walks’ lagging strand also in the 5’ to 3’
along it, adding new complementary direction.
nucleotide bases (A, C, G and T) to
the strand of DNA in the 5’ to 3’ 7. This type of replication is called
direction. discontinuous as the Okazaki
fragments will need to be joined up
7. This sort of replication is called later.
continuous.
35
The Chemistry of
DNA replication
DNA Synthesis by DNA polymerase
8. Once all of the bases are matched up (A with T, C with G), an
enzyme called exonuclease strips away the primer(s). The gaps
where the primer(s) were are then filled by yet more
complementary nucleotides.
9. The new strand is proofread to make sure there are no mistakes in
the new DNA sequence.
10.Finally, an enzyme called DNA ligase seals up the sequence of
DNA into two continuous double strands.
11.The result of DNA replication is two DNA molecules consisting of
one new and one old chain of nucleotides. This is why DNA
replication is described as semi-conservative, half of the chain is
part of the original DNA molecule, half is brand new.
12.Following replication the new DNA automatically winds up into a
double helix.
DNA Proofreading
Biochemistry 2/e - Garrett & Grisham

DNA Replication at the

Lagging strand

Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham

DNA Ligase

Copyright © 1999 by Harcourt Brace & Company

 Termination
• The "ter" locus, rich in Gs and Ts, signals
the end of replication. A Ter protein is
also involved.
• Ter protein is a contrahelicase and
prevents unwinding
Mechanism of Termination:
 Biochemistry 2/e - Garrett & Grisham
Telomeres problem in Eukaryotic
DNA Replication

• Synthesizing the ends of the chromosomes is difficult

because of the lack of a primer.

• Because DNA polymerase cannot complete synthesis of

the 5′ end of each strand.

• With each round of DNA replication, the linear eukaryotic

chromosome becomes so short that the chromosomes
cannot function properly and the cells die.

48
Copyright © 1999 by Harcourt Brace & Company
 Biochemistry 2/e - Garrett & Grisham

Blue color indicates

newly polymerized
nucleotides

49
Copyright © 1999 by Harcourt Brace & Company
 Biochemistry 2/e - Garrett & Grisham

Synthesis of Telomeres in Eukaryotes

• Telomeres – repeated DNA sequence on the ends of eukaryotic
chromosomes

– produced by telomerase

• Telomerase contains an RNA region that is used as a template, as

well as telomerase reverse transcriptase activity (hTRT) so a DNA
primer can be produced.

• Normally telomerase activity is present only in embryonic cells,

germ (reproductive) cells, and stem cells, but not in somatic cells.

• Cancer cells often have relatively high levels of telomerase,

preventing the telomeres from becoming shortened and contributing
to the immortality of malignant cells.

50
Copyright © 1999 by Harcourt Brace & Company
 Biochemistry 2/e - Garrett & Grisham

Telomerase Structure

Reverse transcriptase
with RNA template to
bind to DNA strands

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Biochemistry 2/e - Garrett & Grisham
Telomerase and its function

Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham

53
Copyright © 1999 by Harcourt Brace & Company
Differences between replication in prokaryotes and eukaryotes
Prokaryotic DNA Replication: Eukaryotic DNA Replication:

1. It occurs inside the cytoplasm. 1. It occurs inside the nucleus.

2. There is single origin of replication. 2. Origin of replications are numerous.

3. DNA polymerase III carries out both initiation and 3. Initiation is carried out by DNA polymerase α while
elongation. elongation by DNA polymerase δ and ε.

4. DNA repair and gap filling are done by DNA

polymerase I. 4. The same are performed by DNA polymerase β.

5. RNA primer is removed by DNA polymerase I. 5. RNA primer is removed by DNA polymerase β.

6. Okazaki fragments are large, 1000-2000 6. Okazaki fragments are short, 100-200 nucleotides
nucleotides long. long.

7. Replication is very rapid, some 2000 bp per second. 7. Replication is slow, some 100 nucleotides per
second.

8. DNA gyrase is needed. 8. DNA gyrase is not needed.

9. Have no ends to synthesize 9. Having distinct process for replicating the

telomeres at the ends of their chromosomes

10. Occurs almost continuously 10. Occurs only during the S-phase of the cell cycle
 Inhibitors of DNA replication
• Dauromycin & Adriamycin: interfering with passage of both DNA
and RNA polymerases, thus inhibit both replication and
transcription, antibiotic.
• Actinomycin D: intercalating agent, inhibit both replication and
transcription (antibiotic, anticancer agent).
• Ethidium bromide & proflavin: intercalating agent, inhibit both
replication and transcription.
• Novobiocin & oxolinic acid: Prokaryotic DNA gyrases inhibitor,
antibiotic.
• Aphidicolin: inhibit DNA pol α, δ and ε.
• Rifamycin: inhibit RNA pol, so RNA primer will not available. Inhibit
RT also. (antibiotic, antitumor agent)
 Quinolones and DNA Gyrase
• Quinolones and fluoroquinolones inhibit DNA gyrase (prokaryotic
topoisomerase II), preventing DNA replication and transcription.

• These drugs, which are most active against aerobic gram-negative

bacteria, include:

• Levofloxacin

• Ciprofloxacin

• Moxifloxacin

• Resistance to the drugs has developed over time; current uses

include treatment of gonorrhea and upper and lower urinary tract
infections in both sexes.
Nucleotide analogues and HIV treatment

• One chemotherapeutic treatment of HIV is the use of

AZT (3′-azido-2′,3′-dideoxythymidine) or structurally
related compounds (ddC, and ddI).

• Once AZT enters cells, it can be converted to the

triphosphate derivative and used as a substrate for the
viral reverse transcriptase in synthesizing DNA from its
RNA genome.

• The replacement of an azide instead of a normal

hydroxyl group at the 3′ position of the deoxyribose
prevents further replication by effectively causing chain
termination.
Thank you

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