Lecture 3.part.1Denaturation,Renaturation of DNA Replication of DNA

Denaturation,Renaturation of DNA
Replication of DNA
MOLECULAR BIOLOGY & GENETICS
Qurat-ul-Ain, B. Pharm., M. Phil., Ph.D.,
Visiting Assistant Professor
Department of Physical Therapy, Faculty of Health& Medical Sciences,
Hamdard University, Karachi, Pakistan
Assistant Professor
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Hamdard
University, Karachi, Pakistan.
Qurat-ul-Ain, B. Pharm., M. Phil., Ph.D., qurat.fophu@yahoo.com

1. Denaturation
- the double helix can be denatured in different ways:
heat, pH (acid & alkali),
-chemicals such as urea (poorly), formamide,
dimethylsulfoxide, etc., and by proteins (single-strand
DNA or RNA binding proteins)
- dissociation by heating is also named melting
- progress of denaturation can be monitored by UV
absorption spectroscopy
- hyperchromicity effect : disruption of the stacking
=> 30 to 40 % increase of UV (260 nm) absorption

- Tm : melting temperature
- position in melting profile where 50% is single-stranded
- pseudo-monomolecular reaction: strands are not physically
separated (but A-T rich zones 'melt' first) (dynamic equilibrium)
=> ' denaturation is concentration-independent'
- linear relationship between Tm and %G+C of a duplex
- physical separation requires temperatures far above Tm
- upon fast chilling (e.g. placing the tube in ice-water!) : nucleic
acid remains single-stranded
- slow cooling is necessary to enable the base-pairs (and
stacking) to rebuild
=> 'renaturation requires energy ! '

Partial denaturation : electronmicroscopical analysis of A+T rich regions.

5
DNA Denaturation

G+C Content Is Proportional to Tm
70 80 90 100
1.4
1.2
1.0
Relative
absorbance
(260
nm)
Temperature (C)
Pneumococcus
(38%) G+C E. coli (52%)
S. Marcescens
(58%)
M. Phlei
(66%)

Renaturation
- renaturation is NOT simply the reverse of denaturation
- collision of complementary strands required
- nucleic acid strands are negatively charged in the phosphate moiety
a -1 charge per nucleotide => repulsion
=> hence : requires shielding to allow strands to approach
one another (use of Na+ or K+ salts)
- four parameters in renaturation kinetics
1) concentration of cations
2) incubation temperature (usually 20 to 25 °C below Tm)
3) DNA concentration (related to complexity of the DNA : see below)
4) size of the fragments

DNA denaturation and renaturation : strategic aspects
native DNA
fast chilling
slow chilling
very limited renaturation
(palindromes?)
almost complete renaturation

9
Reversible denaturation and annealing (renaturation) of DNA

GENE

GENE EXPRESSION

DNA Replication
• DNA Replication is the
process in which the
DNA within a cell makes
an exact copy of itself.

It has not escaped our notice
that the specific pairing we have
postulated suggests a possible
copying mechanism for the
genetic material.

• Watson & Crick (1953) - proposed DNA
structure & suggested how it might "self-
duplicate"
• “It has not escaped our notice that the
specific pairing we have postulated
immediately suggests a possible copying
mechanism for the genetic material.”
– A. Suggested that replication occurred by
gradual double helix strand separation via
successive breakage of H bonds, much
like the separation of the two halves of a
zipper
– B. Since each strand is complementary to
the other, each has the information
needed to construct the other; once
separated, each strand can serve as
template to direct the formation of the
other strand
DNA Replication…

Enzymes in DNA Replication

Elongation
• DNA polymerase uses each strand as a template in the 3’ to 5’ direction
to build a complementary strand in the 5’ to 3’ direction
 results in a leading strand and a lagging strand
DNA Replication

Leading Strand
1. Topisomerase unwinds DNA and then Helicase breaks H-bonds
2. DNA primase creates a single RNA primer to start the replication
3. DNA polymerase slides along the leading strand in the 3’ to 5’ direction
synthesizing the matching strand in the 5’ to 3’ direction
4. The RNA primer is degraded by RNase H and replaced with DNA nucleotides by
DNA polymerase, and then DNA ligase connects the fragment at the start of the
new strand to the end of the new strand (in circular chromosomes)
DNA Replication

Lagging Strand
1. Topisomerase unwinds DNA and then Helicase breaks H-bonds
2. DNA primase creates RNA primers in spaced intervals
3. DNA polymerase slides along the leading strand in the 3’ to 5’ direction
synthesizing the matching Okazaki fragments in the 5’ to 3’ direction
4. The RNA primers are degraded by RNase H and replaced with DNA nucleotides
by DNA polymerase
5. DNA ligase connects the Okazaki fragments to one another (covalently bonds the
phosphate in one nucleotide to the deoxyribose of the adjacent nucleotide)
DNA Replication

Topoisomerase - unwinds DNA
Helicase – enzyme that breaks H-bonds
DNA Polymerase – enzyme that catalyzes connection of nucleotides to form complementary
DNA strand in 5’ to 3’ direction (reads template in 3’ to 5’ direction)
Leading Strand – transcribed continuously in 5’ to 3’ direction
Lagging Strand – transcribed in segments in 5’ to 3’ direction (Okazaki fragments)
DNA Primase – enzyme that catalyzes formation of RNA starting segment (RNA primer)
DNA Ligase – enzyme that catalyzes connection of two Okazaki fragments
DNA Replication

Figure 2.3
DNA Replication Animation
Please note that due to differing
operating systems, some animations
will not appear until the presentation is
viewed in Presentation Mode (Slide
Show view). You may see blank slides
in the “Normal” or “Slide Sorter” views.
All animations will appear after viewing
in Presentation Mode and playing each
animation. Most animations will require
the latest version of the Flash Player,
which is available at
http://get.adobe.com/flashplayer.

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

• Possible types of DNA
replication
– 1. Semiconservative - daughter
duplex made of one parental &
one newly synthesized strand
– 2. Conservative - 2 original
strands stay together after serving
as templates for 2 new strands
that also stay together; one
contains only "old" DNA, the other
only "new" DNA
– 3. Dispersive – integrity of both
parental strands disrupted; new
duplex strands made of old & new
DNA; neither the parental strands
nor the parental duplex is
preserved
DNA Replication

Figure 2.3
Experiment Animation
Please note that due to differing
operating systems, some animations
will not appear until the presentation is
viewed in Presentation Mode (Slide
Show view). You may see blank slides
in the “Normal” or “Slide Sorter” views.
All animations will appear after viewing
in Presentation Mode and playing each
animation. Most animations will require
the latest version of the Flash Player,
which is available at
http://get.adobe.com/flashplayer.

• The result
• The result was clean as a whistle. At the start of the experiment
before shifting to the regular medium, they saw only one band of
DNA on the pictures of their density gradient. It consisted exclusively
of heavy DNA as no growth in the regular medium had occurred yet.
After one bacterial generation they still only observed one band
although at a lower density in the gradient (i.e. higher up!), about
half way between control heavy and control light DNA! This means
that DNA replication cannot be conservative, as conservative
replication would lead to two distinct bands, one consisting
exclusively of heavy DNA and one exclusively of light DNA. But that
still leaves options 2 and 3. Yet another round of bacterial growth
gave the final answer: They now saw two distinct bands, one at the
density of the control light DNA and one at the intermediate density
from last round. This excludes dispersive replication as a
mechanism as dispersive replication would have resulted in yet
another single band at a density between the control light DNA and
the control heavy DNA.

DNA Replication
Meselson and Stahl concluded that the
mechanism of DNA replication is the
semiconservative model.
Each strand of DNA acts as a template for
the synthesis of a new strand.

DNA Replication…
• Reproduction is fundamental to all living systems
• Regardless of the reproductive mechanism
(asexual or sexual) a method must exist to
transfer genetic material from one generation to
the next.
• DNA must be copied (replicated) in a manner
that minimizes mistakes.
• Damage to DNA must be repaired to prevent
that damage from being transferred to the next
generation.

Lecture 3.part.1Denaturation,Renaturation of DNA Replication of DNA

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Lecture 3.part.1Denaturation,Renaturation of DNA Replication of DNA