DNA Replication

Before a Cell Divides DNA Must be Replicated

 Unlike other molecular processes (i.e. protein

synthesis) that take place all the time inside the
cell, DNA is replicated only at a particular stage of
the “cell cycle”, JUST BEFORE CELL DIVISION
DNA replication Is Semi-Conservative
The Two Strands Are Antiparallel
 How Is DNA Replicated?
 A replication complex that includes a number of
enzymes, is necessary to catalyze the reactions of
replication.
 All chromosomes have a base sequence called
origin of replication (ori).
 Replication complex binds to ori at start.
 DNA replicates in both directions, forming two
replication forks.
 Each entire chromosome is replicated, unlike
transcription that copies only one gene.
Prokaryotes Have One Origin of Replication
Eukaryotes Have Multiple Origins of Replication
 Replication Complex
 The replication complex includes a number of
proteins:
 Helicase: separates the two DNA strands
 Topoisomerase: swivels
 ssDNA binding proteins: keep the the strands separated
 DNA polymerase: catalyzes the formation of a covalent
bond between the 3’ end of a nucleotide and the 5’
end of the other.
 Primase (an RNA polymerase): synthesizes an RNA
primer
Topisomerase Untangles Supercoils,
ssDNA Binding Proteins Hold the Two
Strands Separated
DNA Polymerase Catalyzes the Formation of
Covalent Bonds Between Nucleotides

Each New DNA Strand Grows from Its 5′ End to Its 3′ End
 Primase and Primers
 A primer is required to start DNA
replication—a short single strand of RNA.
 Primer is synthesized by primase.
 Then DNA polymerase begins adding
nucleotides to the 3′ end of the primer.
NOTE: DNA polymerase moves in the 5’
to 3’ direction of the growing DNA strand
Primase Synthesizes and RNA Primer
 How Is DNA Replicated?

 At the replication fork:

 The leading strand is pointing in the
“right” direction for replication.
 The lagging strand is in the “wrong”
direction.
 Synthesis of the lagging strand occurs in
small, discontinuous stretches—Okazaki
fragments.
The Two New Strands Form in Different Ways
Ligase catalyzes this reaction
Telomeres
 The Process of Life (Eukaryotes):
Cell Division, Ageing and Death

 For most cells this process will not go on forever

(most cells are not immortal)
 Cells grown in cell culture will stop dividing, age
and die after ~ 50 divisions.
 Bacteria are different. They can divide forever.
They will die of causes such as starvation,
poisoning or mechanical insult
The Ends of the Chromosomes Have
Special Sequences Called Telomeres

• Telomeres do not contain genes but repetitions (between 100 and

1000) of one short DNA sequence. TTAGGG (human). Telomeres
prevent important sequences from being eroded.
• Protozoa have it, fungi have it, plants have it, everybody seems to
have it, except prokaryotes, WHY?
 Chromosomes Get Shorter with
Every Round of Cell Division

When non-coding telomere sequences run out, shortening of the

chromosomes will lead to the erosion of genetic material

Erosion of coding sequences leads to cell’s ageing and death

 Limits to Cell Division
 In stem cells (adult and embryonic), and precursors of
germ cells, an enzyme called telomerase restores the
chromosomes endings at every division. These cells
are called immortal.
 Unfortunately cancer cells are also immortal…
 discussed on Friday
Why?

Dolly died at the young age of 6.

Her telomeres were much shorter
than what expected for her age.
The end-replication problem
Why are bacteria “immortal”?
Telomerase
 Turning OFF the Telomerase Gene
 The telomerase enzyme is composed of an RNA subunit and a
proteic subunit.
 The gene TEP1 codes for the proteic subunit .
 TEP 1 is active in every cell in the early embryo.
 As the embryo develops, the genes that make telomerase are
switched off in all but a few tissues. These tissues are the ones
that will give rise to the reproductive cells and other adult stem
cells
 From the moment telomerase is shut off, the effect is like
setting a stopwatch and telomeres length declines by 50 to 100
bp each generation. When telomeres become too short cells
reach a point of senescence.
 There is variation among people in telomere length. From 7000
bases to about 10,000. Telomere length is heritable as is
longevity.
Relationship between mean (± SEM) telomere length (T/S ratio from qPCR) and age at measurement
(first sample, shown as year = 0, was collected at 25 d) in zebra finches in three lifespan categories.

Britt J. Heidinger et al. PNAS 2012;109:1743-1748

©2012 by National Academy of Sciences

Effect of comprehensive lifestyle changes on telomerase activity and telomere length in men
with biopsy-proven low-risk prostate cancer: 5-year follow-up of a descriptive pilot study

(diet, activity, stress management, and social support)

The Lancet Oncology 2013 14, 1112-1120DOI: (10.1016/S1470-2045(13)70366-8)

Copyright © 2013 Elsevier Ltd Terms and Conditions
Dr. K Mullis: The Polymerase
Chain Reaction (PCR)
 PCR Amplifies Any DNA Sequence of
Approximately 1 to 2 thousands Base Pairs
 Paleobiology
 Extinct species
 Human migration
 Sequencing
 Genomes and individual genes
 Basic research
 Medicine, genetic conditions, infections
 Forensic
 Cloning
 Replicating DNA in a Test Tube:
The Polymerase Chain Reaction

“Ingredients”

Heat stable
 Where Do the “Ingredients”
Come From?
 Template is the genomic DNA you will extract from
bacteris
 Primers are designed to flank the region of interest
and chemically synthesized
 Taq Polymerase is islolated by the archaon
Thermus acquaticus
 How to Design Primers to
Amplify a Known Sequence
Reverse Primer

5’ AGC TTA CGC AAC CGC TAC AGT TTC AGG CTA GCA TAG CAT GGA CTA CCA ATG 3’

Polymerization 3’ GAT GGT TAC 5’

5’ AGC TTA CGC Polymerization

3’ TCG AAT GCG TTG GCG ATG TCA AAG TCC GAT CGT ATC GTA CCT GAT GGT TAC 5’

Forward Primer

In RED is the region we want to amplify

NOTE: PRIMERS ARE LONGER, 18-25 BASES LONG
Small oligonucleotides to be used as primers can be chemically synthesized.
 The Beta Subunit Gene
of Bacterial RNA Polymerase

Reverse primer

Forward primer

We will amplify this region because research has shown that this
is where mutations occur that confer resistance to rifampicin.
Comparison with DNA Replication in a Cell

98 0C
Breaks hydrogen
bonds

60-65 0C
Anneals
the primers

72 0C
Ideal for Taq
polymerase
activity
Replicating DNA in a Test Tube:
The Polymerase Chain Reaction
https://www.youtube.com/wa
tch?v=_YgXcJ4n-
kQ&feature=kp