DNA structure, and the bonds that stabilizes it. The structural components, units and the proteins involved. Types of DNA and its separation methods. Chargaffs rule and its application
2. Outline
Terms used.
Polarity
Bonds present
Proteins involved
Properties of nucleosides and nucleotides
Structure of DNA
Watson Crick model
Types of DNA
DNA sequencing
3. Terminologies….
Nitrogenous base.
Nitrogen containing.
Free pyrimidines and purines are weakly basic
compounds and thus are called bases
Sugar
2-Deoxy D-ribose pentose sugar
4.
5. Terminologies….
Nucleoside
nitrogenous (nitrogen-containing) base
a pentose
Nucleoside with
Purine base have suffix –sine
Pyrimidine base have suffix –dine
Nucleotide
nitrogenous (nitrogen-containing) base
a pentose
a phosphate
Nucleic acid
Linear polymers of nucleotides
6. Introns
Non-coding, intervening , silent areas
Exons
Coding, expressed regions
Cistrons
Unit of gene expression, biochemical counterpart of gene
Gene
A segment of a DNA molecule that contains the
information required for the synthesis of a functional
biological product, whether protein or RNA.
11. Polarity
In DNA the base sequence is always written in 5I end
to 3I end direction
By definition, the 5 end lacks a nucleotide at the 5
position and the 3 end lacks a nucleotide at the 3
position.
5I -OH and 3I- OH ends.
The 5 to 3 orientation of a strand of nucleic acid
refers to the ends of the strand.
12. Bonds involved…
N-9 of a purine or N-1 of a pyrimidine is attached to
C-1 of the sugar
The base lies above the plane of sugar when the
structure is written in the standard orientation;
The configuration of the N-glycosidic linkage is β .
13. Bonds involved…
The successive nucleotides of DNA covalently linked
through phosphate-group “bridges”
5-phosphate group of one nucleotide unit is joined to
the 3-hydroxyl group of the next nucleotide
3I- to 5I Phosphodiester linkage
14.
15. Covalent backbones of nucleic acids consist of
alternating phosphate and pentose residues,
Nitrogenous bases as side groups joined at regular
intervals.
Linkages can be cleaved hydrolytically by chemicals
or enzymatically by family of Nucleases.
16. Comments…
The covalent backbone of DNA and RNA is subject
to slow, non-enzymatic hydrolysis of the
phosphodiester bonds.
In the test tube, RNA is hydrolyzed rapidly under
alkaline conditions but DNA is not.
17. Hydrogen bonds
Involving the amino and carbonyl groups are the
most important mode of interaction between two
complementary strands of nucleic acid.
Required for specificity of base pairing
18.
19. Other bonds…
The stacking also involves a combination of van der
Waals and dipole-dipole interactions between the
bases.
Base stacking helps to minimize contact of the bases
with water, and
Base -stacking interactions are very important in
stabilizing the three dimensional structure of nucleic
acids
Base stacking in DNA is also favored by the
conformations of the relatively rigid five-membered
rings of the backbone sugars.
The sugar rigidity affects both the single-stranded
and the double-helical forms.
20.
21. Properties of nucleosides and nucleotides
Physical properties
Charged phosphate groups provide interaction.
Electron delocalization among atoms in the ring
gives most of the bonds partial double-bond
character.
Strong absorption at wavelengths near 260 nm.
Purines have higher molar extinction coefficient.
22.
23. Structural properties
Sugar puckering
Displacement of 2 and 3 carbon atom above the
plane of C1-O4-C4
By convention above is the direction in which the
base and C5I project from the ring and is termed the
endo face of the pentose.
If C2 is above it is called C2 endo
If C3 is above it is called C3 endo
Orientation of glycosidic bond and the distance of
the phosphate bond changes.
DNA: C2 endo and RNA C3 endo.
24.
25. Anti- & Syn- conformation…
Nucleosides are planar.
Still free rotation of glycosidic bond is possible.
In purines
H8 above sugar- anti conformation
H8 below and far with bulk og purine ring above sugar-
syn
In pyrimidines in pyrimidines
H6 is above sugar- anti conformation
O2 is above sugar- syn conformation
26.
27.
28. Comments…
Purines favour anti conformation..
Interconvert between the two
Guanine 5I nucleotides is always an exception
Favours syn conformation.
Importance
Defines the handedness of the DNA helix.
Anti conformation- right handed helix
Syn- conformation- left handed helix
DNA having more of G will have left handed helix
29. Handedness
If you look up through the
bottom of a helix along the
central axis and the helix
spirals away from you in a
clockwise direction it is a
right-handed helix.
If it spirals away from you
in a counter-clockwise
direction, it is a left
handed helix.
30. Proteins involved…
Histones
Genes for histones seem to have no introns
Nucleosome
Dna wraps twice around histone octamer
32. Condensed Chromosome Structures Are
Maintained by….
A third major class of chromatin proteins SMC
proteins (structural maintenance of chromosomes).
Proteins in the SMC family are found in all types of
organisms, from bacteria to humans.
Eukaryotes have two major types, cohesins and
condensins.
Cohesins
linking together sister chromatids
Condensins –
condensation of chromosomes as cells enter mitosis
33.
34. DNA & its history…
DNA is in nucleus..
Storage of inorganic phosphate..
It carries genetic information
It is passed to daughter cell…
Then
Experiments to elucidate the structure..
35. DNA helix in history….
DNA was first isolated and characterized by Friedrich
Miescher in 1868.
He called the phosphorus-containing substance
“nuclein.”
Alfred D. Hershey and Martha Chase, in which they
studied the infection of bacterial cells by a virus
(bacteriophage) with radioactively labeled DNA or
protein, removed any remaining doubt that DNA, not
protein, carried the genetic information
36. 1951—Rosalind Franklin—X-ray crystallography
Chargaff—Chargaff’s rules.
Ratio of nitrogenous bases in DNA.
Complimentary bases.
Watson & Crick--1953
39. Chargaff’s conclusions
Base composition of DNA varies from one species
to another.
DNA specimens isolated from different tissues of the
same species have the same base composition.
The base composition of DNA in a given species
does not change with an organism’s age, nutritional
state, or changing environment.
In all cellular DNAs, regardless of the species,
Number of adenosine residues is equal to thymidine
residues (that
Number of guanosine residues is equal to cytidine
residues
Sum of the purine residues equals pyrimidine
residues
41. Two helical polynucleotide chains are coiled around
a common axis. The chains run in opposite
directions.
The sugar-phosphate backbones are on the outside
and, therefore,
Purine and pyrimidine bases lie on the inside of the
helix.
The bases are nearly perpendicular to the helix axis,
and adjacent bases are separated by 3.4 Å.
The helical structure repeats every 34 Å, so there
are 10 bases (= 34 Å per repeat/3.4 Å per base) per
turn of helix.
42. There is a rotation of 36 degrees per base (360
degrees per full turn/10 bases per turn).
The diameter of the helix is 20 Å.
The complimentary nitrogenous bases form
hydrogen bonds between the strands.
A is complimentary to T and G is complimentary to
C.
43. B- DNA, right handed, 10 base pairs per turn.
Two chains coiled around a common axis: Axis of
symmetry.
Pairs in an anti- parallel manner i.e. 5′- end pairs
with 3′- end of other strand.
Phosphate molecules are hydrophillic, whereas
bases forms the hydrophobic molecules.
44. Spatial arrangement of two strands creates Major
(wide) & minor (narrow) Groove.
Bases are perpendicular to axis, sugars are at right
angle to those of bases.
Diameter of helix is 20nm, adjacent bases
separated by 3.4Ǻ.
One complete turn of 10 BP occurs at interval of
34Ǻ.
45.
46.
47.
48.
49.
50. Circular DNA molecules
Each chromosome in the nucleus of a eukaryote
contains one long linear molecule of dsDNA.
Eukaryotes have closed circular DNA molecules in
their mitochondria.
A prokaryotic organism contains a single, double-
stranded, supercoiled, circular chromosome.
Each prokaryotic chromosome is associated with
histone-like proteins and RNA that can condense the
DNA to form a nucleoid.
Species of bacteria also contain small, circular,
extrachromosomal DNA molecules called plasmids.
51. Other types of DNA
Bent DNA
4-6 adenines separated by 10 bp.
Minor grooves compressed
Interaction with proteins & enzymes
Cruciform DNA
Disruption of hydrogen bonds between complimentary bp
Formation of intra strand hydrogen bonds.
Formation of hairpin
52. Other types of DNA
Triple stranded DNA
Polynucleotides of Poly (dA) & poly (dT)
Hoogsteen triple helix
TAT, CGC triplet bp
Four stranded DNA
Rich in guanine nucleotides.
G-quartets.
Guanine with hoogsteen hydrogen bonds.
Existence invivo not proven
53. Slipped DNA
Direct repeat symmetry
Formation of two single stranded loops.
Fragile X syndrome typical example of triple repeat
sequence.
Frame shift mutation explained on this basis.
56. Melting temperature
Temperature at which one half of the helical structure
is lost is defined as the melting temperature (Tm).
The loss of helical structure in DNA, called
denaturation, can be monitored by measuring its
absorbance at 260 nm
Ss DNA has a higher relative absorbance at this
wavelength than does ds DNA.
Complementary DNA strands can reform the double
helix by the process called renaturation (or
reannealing).
57.
58.
59.
60. Importance of Tm
Critical importance in any technique that relies on
complementary base pairing
Designing PCR primers
Southern blots
Northern blots
Colony hybridization
61. Factors Affecting Tm
G-C content of sample
Presence of intercalating agents (anything that
disrupts H-bonds or base stacking)
Salt concentration
pH
Length
63. DNA Sequencing
1. Sanger dideoxynucleotide chain termination method
A. Manual method
B. Automated method
2. Chemical cleavage method (Maxam and Gilbert
method)
Not used nowadays
Use of the technique
Provides the order of the nucleotides in a given DNA
64. Sanger Method
Partial copies of DNA fragments made with DNA
polymerase
Collection of DNA fragments that terminate with
A,C,G or T using ddNTP
Separate by gel electrophoresis
Read DNA sequence
65. Requirements for Sanger Method
DNA to be sequenced must be in single strand form.
The region to be sequenced must be 3I flanked by
known sequence.
Reagents needed:
A primer complementary to the known region to start and
direct chain synthesis. (15-30 nucleotides in length)
DNA polymerase.
4 deoxynucleotide triphosphates (dNTPs).
4 dideoxynucleotide triphosphates (ddNTPs)
66.
67.
68.
69.
70.
71. Take home message…
All life on earth use nucleic acid to store genetic
information.
Except some viruses(RNA) it is stored in DNA.
So why DNA???
Chemical stability..
Store vast genetic information
And most important!!!!!
Encode this vast genetic information with simple four
letter code(A,G,C &T)
72. References..
Text Book Of Biochemistry With Clinical Correlation
By Thomas Devlin 7th Edition
Lehninger Principles Of Biochemistry. 5th Edition
Lippincott's Illustrated Reviews: Biochemistry 4th
Edition
Stryer's Biochemistry- 5th edition
Marks’ Basic Medical Biochemistry: A Clinical
Approach, 2nd Edition
Textbook Of Biochemistry DM Vasudevan 6th Edition
