DNA,REPLICATION,POLYMERASES,SEMICONSERVATIVE

1. Molecular Genetics
The Central Dogma of Biology
Sardar Hussain
Asst.prof.biotechnology,
Gsc.cta.
Sardar1109@gmail.com

2. What is a Genetic “Factor”?
• From Mendel:
– we now accepted that there was genetic
transmission of traits.
• Traits are transmitted by “factors”
– Organisms carry 2 copies of each “factor”
• The question now was: what is the factor
that carries the genetic information?

3. Requirements of Genetic Material
• Must be able to replicate, so it is reproduced in each
cell of a growing organism.
• Must be able to control expression of traits
– Traits are determined by the proteins that act within us
– Proteins are determined by their sequences
• Therefore, the genetic material must be able to
encode the sequence of proteins
• It must be able to change in a controlled way, to
allow variation, adaptation, thus survival in a
changing environment.

4. Chromosomes – The First
Clue
• First ability to visualize chromosomes in the
nucleus came at the turn of the century
– construction of increasingly powerful
microscopes
– the discovery of dyes that selectively colored
various components of the cell
• Scientists examined cellular nuclei and
observed nuclear structures, which they
called chromosomes
• Observation of these structures suggested
their role in genetic transmission

5. Chromosome Observations
• Variety of chromosome types found in the nucleus
• 2 copies of each type of chromosome in most cells.
• All of the cells of an organism, except gametes and
rbc’s, have the same number of chromosomes.
• All organisms of the same species have the same
number of chromosomes.
• The number of chromosomes in a cell doubles
immediately prior to cell division
• Gametes have exactly half of the number of
chromosomes as the somatic cells of any organism.
– Gametes have just one copy of each chromosome type.
– Fertilization produces a diploid cell (a zygote), with the same
number of chromosomes as somatic cells of that organism.

6. Implications
• Chromosomes behaved like Mendel’s “factors”
– Mendel's hereditary factors were either located on
the chromosomes or were the chromosomes
themselves.
• Proof chromosomes were hereditary factors – 1905:
• The first physical trait was linked to the presence of
specific chromosomal material
– conversely, the absence of that chromosome meant
the absence of the particular physical trait.
• Discovery of the sex chromosomes
– "X" and "Y."
– distinguished from other chromosomes and from
each other

7. Importance of Sex Chromosomes
• Somatic cells taken from female donors always
contained 2 copies of the X chromosome
• Somatic cells taken from male donors always
contained one copy of the X chromosome and one
copy of the Y chromosome
• All of the other chromosomes in the nucleated cells
of both male and female donors appeared identical
• Thus, gender was shown to be the direct result of a
specific combination of chromosomal material
– The first phenotype (physical characteristic) to be
assigned a chromosomal location
– Specifically the X and Y chromosomes.

9. What Carries the Genetic Information?
• Chromosomes are about 40% DNA & 60% protein.
– Protein is the larger component
• Protein molecules are composed of 20 different
subunits
• DNA molecules are composed of only four
• Therefore protein molecules could encode more
information, and a greater variety of information
– protein had the possibility for much more diversity than in
DNA
• Therefore, scientists believed that the protein in
chromosomes must carry the genetic information

10. The Discovery of DNA
• First identified in 1868 by Friedrich
Miescher, a Swiss biologist, in the
nuclei of pus cells obtained from
discarded surgical bandages.
• He called the substance nuclein
• Noted the presence of phosphorous

11. The Transforming Principle
• Fredrick Griffith - 1928
• Discovered that different strains of the bacterium
Strepotococcus pneumonae had different effects on
mice
– One strain could kill an injected mouse (virulent)
– Another strain had no effect (avirulent)
– When the virulent strain was heat-killed and injected into
mice, there was no effect.
– But when a heat-killed virulent strain was co-injected with the
avirulent strain, the mice died.
• Concluded that some factor in the heat killed bacteria
was transforming the living avirulent to virulent?
• What was the transforming principle and was this the
genetic material?

13. The Transforming Principle is DNA
• Avery, Macleod, & McCarty – 1943
• Attempted to identify Griffith’s “transforming
principle”
• Separated the dead virulent cells into fractions
– The protein fraction
– DNA fraction
• Co-injected them with the avirulent strain.
– When co-injected with protein fraction, the mice lived
– with the DNA fraction, the mice died
• Result was IGNORED
– Most scientists believed protein was the genetic material.
– They discounted this result and said that there must have
been some protein in the fraction that conferred virulence.

14. The Hershey-Chase Experiment
• Hershey & Chase – 1952
• Performed the definitive experiment that
showed that DNA was the genetic
material.
• Bacteriphages = viruses that infect
bacteria
• Bacteriphage is composed only of
protein & DNA
• Inject their genetic material into the host

16. The Experiment
• Prepared 2 cultures of bacteriophages
• Radiolabeled sulphur in one culture
– there is sulphur in proteins, in the amino acids methionine
and cysteine
– there is no sulphur in DNA
• Radiolabeled phosphorous in the second culture
– there is phosphorous in the phosphate backbone of DNA
– none in any of the amino acids.
• So this one culture in which only the phage protein
was labeled, and one culture in which only the phage
DNA was labeled.

17. Experiment Summary
• Performed side by side experiments with separate
phage cultures in which either the protein capsule
was labeled with radioactive sulfur or the DNA
core was labeled with radioactive phosphorus.
• The radioactively labeled phages were allowed to
infect bacteria.
• Agitation in a blender dislodged phage particles
from bacterial cells.
• Centrifugation pelleted cells, separating them from
the phage particles left in the supernatant.

18. Results Summary
• Radioactive sulfur was found predominantly in the
supernatant.
• Radioactive phosphorus was found predominantly
in the cell fraction, from which a new generation of
infective phage was generated.
• Thus, it was shown that the genetic material that
encoded the growth of a new generation of phage
was in the phosphorous-containing DNA.

20. Chargaff’s Rule
• Once DNA was recognized as the genetic material,
scientists began investigating its mechanism and
structure.
• Erwin Chargaff – 1950
– discovered the % content of the 4 nucleotides was the
same in all tissues of the same species
– percentages could vary from species to species.
• He also found that in all animals (Chargaff’s rule):
%G = %C
%A = %T
• This suggested that the structure of the DNA was
specific and conserved in each organism.
• The significance of these results was initially
overlooked

21. Base Pairing in DNA
• Not understood ‘til Watson &
Crick described double helix
• Adenine & guanine are purines
– 2 organic rings
• Cytosine & guanine are
pyrimidines
– 1 organic ring
• Pairing a purine & a pyrimidine
creates the correct 2 nm distance
in the double helix
• A – T joined by 2 hydrogen
bonds
• G – C joined by 3 hydrogen
bonds

22. The Double Helix
• James Watson and Francis Crick – 1953
• Presented a model of the structure of DNA.
• It was already known from chemical studies
that DNA was a polymer of nucleotide
(sugar, base and phosphate) units.
• X-ray crystallographic data obtained by
Rosalind Franklin, combined with the
previous results from Chargaff and others,
were fitted together by Watson and Crick
into the double helix model.

23. Watson and Crick shared the 1962 Nobel Prize for
Physiology and Medicine with Maurice Wilkins.
Rosalind Franklin died before this date.

24. DNA Structure
• The double helix is formed from two strands of DNA
• DNA strands run in opposite directions
• They are complementary
– attached by hydrogen bonds between complimentary
base pairs:
– A - T and G - C
• This complementary pairing of the bases suggests
that, when DNA replicates, an exact duplicate of the
parental genetic information is made.
– The polymerization of a new complementary strand takes
place using each of the old strands as a template.

26. Messelson and Stahl
• How does DNA replicate?
• Matthew Meselson and Franklin Stahl - 1957
• Did an experiment to determine which model best
represented DNA replication:
• semiconservative replication
– the two strands unwind and each acts as a template for
new strands
– each new strand is half comprised of molecules from
the old strand
• conservative replication
– the strands do not unwind, but somehow generate a
new double stranded DNA copy of entirely new
molecules

29. The Experiment
• The original DNA strand was labeled with the
heavy isotope of nitrogen, N-15.
• This DNA was allowed to go through one
round of replication with N-14 (non-labeled)
• the mixture was centrifuged so that the
heavier DNA would form a band lower in the
tube
• the intermediate (one N-15 strand and one N-
14 strand) and light DNA (all N-14) would
appear as a band higher in the tube

30. The expected
results for each
model were:

31. The actual results were as expected for the
semiconservative model and thus Watson and
Crick's suspicion was borne out.

32. Enzymes & Replication
• DNA replication is not a passive, spontaneous
process.
• More than a dozen enzymes & other proteins are
required to unwind the double helix & to synthesize
& finalize a new strand of DNA.
• The molecular mechanism of DNA replication can
best be understood from the point of view of the
machinery required to accomplish it.
• The unwound helix, with each strand being
synthesized into a new double helix, is called the
replication fork.

33. The Enzymes of DNA Replication
• Topoisomerase
– Responsible for initiation of unwinding of DNA.
– The tension holding the helix in its coiled and
supercoiled structure is broken by nicking a single
strand of DNA.
• Helicase
– Accomplishes unwinding of the original double
strand, once supercoiling is eliminated by
topoisomerase.
– The two strands want to bind together because of
hydrogen bonding affinity for each other, so
helicase requires energy (ATP) to break the
strands apart.

34. Single-stranded Binding Proteins
• Important to maintain the stability of the
replication fork.
• Line up along unpaired DNA strans,
holding them apart
• Single-stranded DNA is very unstable,
so these proteins bind bind to it while it
remains single stranded and keep it
from being degraded.

35. Beginning: Origins of Replication
• Replication begins at specific sites called origins of
replication
• In prokaryotes, the bacterial chromosome has a
specific origin
• In eukaryotes, replication begins at many sites on
the large molecule
– 100’s of origins
• Proteins that begin replication recognize the origin
sequence
• These enzymes attach to DNA, separating the
strands, and opening a replication bubble
• The end of the replication bubble is the “Y” shaped
replication fork, where new strands of DNA elongate

37. Elongation
• Elongation of the new DNA strands is catalyzed by
DNA polymerase
• Nucleotides align with complimentary bases on the
template strand, and are added by the polymerase,
one by one, to the growing chain
– DNA polymerase proceeds along a single-stranded DNA
molecule, recruiting free nucleotides to H-bond with the
complementary nucleotides on the single strand
• Forms a covalent phosphodiester bond with the
previous nucleotide of the same strand
– The energy stored in the triphosphate is used to covalently
bind each new nucleotide to the growing second strand
• Replication proceeds in both directions

39. The Replication Fork

40. DNA Polymerase
• There are different forms of DNA
polymerase
– DNA polymerase III is responsible for the
synthesis of new DNA strands
• DNA polymerase is actually an aggregate
of several different protein subunits, so it is
often called a holoenzyme.
• Primary job is adding nucleotides to the
growing chain
• Also has proofreading activities

41. Proofreading & Repair
• DNA polymerase proofreads each
nucleotide added against its’ template
as it is added
• Removes incorrectly paired nucleotides
& corrects

42. DNA Strands are Anti-parallel
• The sugar phosphate backbones of the 2
parent strands run in opposite directions
– “upside down” to each other
• DNA is polar
– There is a 3’ end and a 5’ end
– At the 3’ end, a OH is attached to the 3’ C of the
last deoxyribose
– At the other end a phosphate group is attached to
the 5’ C of the last nucleotide
• DNA polymerase adds nucleotides only to the
3’ end of the growing chain
– So new DNA strand elongates only in the 5’ 3”
direction

44. Why 5’  3’?
• Why can DNA polymerase only add nucleotides to
the 3’ end ?
• Needs a triphosphate to provide energy for the
bond between an added nucleotide & the growing
DNA strand.
• This triphosphate is very unstable
– can easily break into a monophosphate and an inorganic
pyrophosphate
• At the 5' end, this triphosphate can easily break
– It is not be able to attach new nucleotides to the 5' end
once the phosphate has broken off
• The 3' end only has a hydroxyl group
– As long as new nucleotide triphosphates are brought by
DNA polymerase, synthesis of a new strand continues,
no matter how long the 3' end has remained free.

45. Leading & Lagging Strands
• The new strand made by adding to the 3’ end =
leading strand
– Parent strand is 3’  5’
– New strand is 5’  3’
• How can DNA polymerase synthesize new copies
of the 5'  3' strand, if it can only travel in one
direction?
• To elongate in the other direction, the process must
work away from the replication fork
• The new strand formed on the 5’  3’ parent strand
is called the lagging strand

46. Building the Lagging Strand
• DNA polymerase makes a second copy
in an overall 3’  5’ direction
• First, it produces short segments,
called Okazaki fragments
– These are built in a 5’ –3’ direction
• Okazaki fragments are joined together
by ligase to produce the new 3’5’
lagging strand

47. Ligase
• Catalyzes the formation of a
phosphodiester bond given an
unattached adjacent 3‘ OH and 5‘
phosphate.
• This can join Okazaki fragments
• This can also fill in the unattached gap
left when an RNA primer is removed
• DNA polymerase can organize the bond
on the 5' end of the primer, but ligase is
needed to make the bond on the 3' end.

49. Primers
• DNA polymerase cannot start synthesizing on
a bare single strand.
– It only adds to an existing chain
• It needs a primer with a 3'OH group on which
to attach a nucleotide.
• The start of a new chain is not DNA, but a
short RNA primer
– Only one primer is needed for the leading strand
– One primer for each Okazaki fragment on the
lagging strand

50. Primase
• Part of an aggregate of proteins called
the primeosome.
• Attaches the small RNA primer to the
single-stranded DNA which acts as a
substitute 3'OH so DNA polymerase
can begin synthesis
• This RNA primer is eventually removed
by RNase H
– the gap is filled in by DNA polymerase I.

52. Ending the Strand
• DNA polymerase only adds to the 3’ end
• There is no way to complete the 5’ end of the new
strand
• A small gap would be left at the 5’ end of each new
strand
• Repeated replication would then make the strand
shorter and shorter, eventually losing genes
• Not a problem in prokaryotes, because the DNA is
circular
– There are no “ends”

53. Telomeres
• Eukaryotes have a special sequence of
repeated nucleotides at the end, called
telomeres
• Multiple repititions of a short nucleitide sequence
– Can be repeated hundreds, or thousands of times
– In humans, TTAGGG
• Do not contain genes
• Protects genes from erosion thru repeated
replication
• Precvents unfinished ends from activating DNA
monitoring & repair mechanisms

54. Telomerase
• Catalyzes lengthening of telomeres
• Includes a short RNA template with the
enzyme
• Present in immortal cell lines and in the
cells that give rise to gametes
• Not found in most somatic cells
• May account for finite life span of
tissues

56. Further Proofreading & Repair
• Some errors evade initial proofreading
or occur after synthesis
• DNA can be damaged by reactive
chemicals, x-rays, UV, etc
• Cells continually monitor DNA for
mutations & repair
• Contain 100’s of repair enzymes

57. Nucleotide Excision & Repair
• A segment of DNA containing damage is cut
out by a nuclease (a DNA cutting enzyme)
• The gap is filled & closed by DNA
polymerase and ligase
• Thymine dimers
• Covalent linking of thymine bases
• Causes DNA to “buckle”
• One common problem corrected this way

59. Proteins of DNA Replication

60. Summary View of Replication