Basics of DNA structure

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Basics of DNA structure

•      DNA: A polymer of deoxyribonucleotides

•      Self-replicating

•      Encodes genetic information in all living organisms and some viruses

•      Found in chromosomes, mitochondria, chloroplast in case of eukaryotes and in nucleoid in case of prokaryotes

•      Gene: DNA segments that encode a functional protein

•      Non-coding regions of DNA have structural and regulatory roles

Nucleotides and Nucleosides

•     Nucleotides are the building blocks of nucleic acids.

•      Nucleotides have three characteristic components:

•      a nitrogenous base

•      a pentose

•      a phosphate

•      The molecule without the phosphate group is called a nucleoside.

•  The nitrogenous bases are derivatives of two parent-compounds: pyrimidine and purine.

Types of bonds in nucleotides

•      Ester bond:

–     Between phosphate and sugar

•      Glycosidic bond:

–     Between sugar and base

 Nomenclature of nucleic acid components

Base	Nucleoside	Nucleotide	Nucleic acid
Purines
Adenine	Adenosine	Adenylate	RNA
	Deoxyadenosine	Deoxyadenylate	DNA
Guanine	Guanosine	Guanylate	RNA
	Deoxyguanosine	Deoxyguanylate	DNA
Pyrimidines
Cytosine	Cytidine	Cytidylate	RNA
	Deoxycytidine	Deoxycytidylate	DNA
Thymine	Thymidine (deoxy)	Thymidylate (deoxy)	DNA
Uracil	Uridine	Uridylate	RNA

Primary structure of DNA

•    DNA consists of two long polymers of deoxyribonucleotides.

• The backbone is formed by deoxyribose (sugar) and phosphate groups linked by phosphodiester bonds.

•  Attached to each sugar is one of the four nitrogenous bases- adenine, guanine, thymine, and cytosine.

•   Traditionally, DNA is drawn from 5’ → 3’ direction.

Secondary structure of DNA

•      DNA is a double-helical structure.

•      The two strands wound around the same axis.

•   The two strands are anti-parallel, i.e. their 5’ →  3’ phosphodiester bonds run in opposite directions.

•      The two strands are complementary to each other.

•      The sugar-phosphate backbone forms the periphery while the bases are stacked in the core.

•      The two strands are held together by hydrogen bonds between two nucleotides in the opposite strands.

•      Adenosine forms two hydrogen bonds with thymidine.

•      Guanosine forms three hydrogen bonds with cytidine.

•      The bases are stacked inside the helix at right angles to the helix axis.

•      The hydrophobic interaction and van der Waals force between stacked bases are also important to maintain the double-helical structure.

•  As the DNA strands wind around each other they leave gaps between each set of phosphate backbone. These grooves are of two types:

–     Major groove (22 Å wide)

–     Minor groove (12 Å wide)

•  The edges of the bases are more accessible in the major groove where proteins (like transcription factors) can interact.

•   The angle at which two sugars protrude from the base pair (i.e. the angle between the glycosidic bonds) is around 120°.

•    As the bases stack on each other, the narrow-angle between the sugars on one edge of the bases generates minor grooves while the large angle on the other edge generates major groove.

•   Edges of each base are exposed at the major and minor groove, creating a pattern of hydrogen bond acceptors and donors and of van der Waals surface that identifies the base pair.

•  These patterns help proteins to specifically recognize DNA sequence without unwinding the double helix.

Types of bonds present in DNA

•      COVALENT BONDS:

–     Ester bond (phosphodiester bond):

•      Between phosphate and sugar

–     Glycosidic bond:

•      Between sugar and base

•      HYDROGEN BOND:

–     Between the base pairs

VAN DER WAALS INTERACTIONS (not within DNA structure): Between major and minor grooves and interacting proteins

Bonds affecting DNA stability

·         HYDROGEN BOND: stabilize

o   Relatively  weak  but additive and facilitates base stacking

o   Hydrogen bond also forms due to the formation of a water spine in the minor groove

·         HYDROPHOBIC INTERACTION: stabilize

o   Hydrophilic groups are oriented outside while hydrophobic groups are outside

·         STACKING INTERACTION: stabilize

o   Relatively  weak  but additive and van der Waals force

o   The π-π between bases in the helical stacks greatly stabilize the double helix

·         ELECTROSTATIC INTERACTION: destabilize

o   Contributed primarily by phosphate groups

o   Affects intra- and inter-strand interactions

o   This repulsive forces can be neutralized by positively charged proteins and  Na⁺ ions

Properties of DNA

UV radiation:

•    DNA absorbs UV rays at 260 nm. When DNA is denatured from double-stranded to single-stranded form, its UV absorbance increases cooperatively.

•      The absorption pattern also increases with an increase in GC content.

•      This is called a hyperchromatic shift.

•      For RNA, the increase in the absorbance pattern is gradual and irregular.

•      DNA absorbs UV due to the aromatic bases.

•      The A₂₆₀ value of the same amount of DNA (e.g 50μg) will differ for single-stranded and double-stranded forms.

–     Double-stranded DNA: A₂₆₀ = 1.00

–     Single-stranded DNA: A₂₆₀ = 1.37

–     Free bases: A₂₆₀ = 1.60

•      The purity of nucleic acids can be estimated by A₂₆₀ / A₂₈₀ values:

–     Pure DNA: A₂₆₀ / A₂₈₀ = 1.80

–     Pure RNA: A₂₆₀ / A₂₈₀ = 2.00

Denaturation:

•   If double-stranded DNA is treated with strong alkali or high temperature, the two strands unwind and separate. This is known as denaturation.

•      The temperature at which the two strands separates is called the melting temperature (T_m) and the process is called the melting of DNA.

Renaturation:

•  The process by which two separated DNA strands form a double helix again is called renaturation.

•      This is done by lowering the temperature to 25°C or below.

•      R.T. Britten and D.E. Kohne (1970) obtained renatured DNA by lowering the temperature and determined its C₀t value.

•      C₀=primary density; t=interval of time in seconds

C₀t curve analysis:

•      It is a technique to determine the complexity of the genome (DNA).

•      The technique is based on the principles of DNA reassociation kinetics.

•      It measures how much repetitive DNA is present in a DNA sample.

•      The technique was developed by R.T. Britten and D.E. Kohne (1970). 

•      Principle:

–  The rate of DNA renaturation is directly proportional to the number of times, the sequences are present in the genome.

–     Given enough time, all denatured DNA will eventually renature.

–     The more the repetitive sequence, the lesser the time required for renaturation.

–   Renaturation also depends on DNA concentration, reassociation temperature, cation concentration, and viscosity.

•   C₀t=DNA conc. (moles/L) x renaturation time (s) x buffer factor (constant for buffer composition and accounts for the effect of cations)

•      C₀=primary density; t=interval of time in seconds

•      Low C₀t value: more repetitive sequences; High C₀t value: more unique and less repetitive sequences.

•      Application:

–     Used for determining genomic complexity in different organisms

–  It can detect the repetitive DNA sequence present in the sample that dominates eukaryotic genomes

–    This allows DNA sequencing to concentrate on the parts of the genome that is most informative and interesting, which will speed up the discovery of new genes and make the process more efficient.

•      Example:

–     Bacterial genome: 99.7% Single copy

–     Calf genome: 17% High repeat, 23% Intermediate repeat, 60% Single copy

Buoyant density:

•   DNA has a buoyant density similar to CsCl. Hence DNA can be mixed with CsCl and separated by centrifugation.

 DNA is acidic:

•     Though nitrogen bases are present in DNA, it is called nucleic acid. This is because these bases are stacked inside the double helix and are not available for interactions. The backbone is formed by sugar-phosphate where the phosphate is negatively charged and has acidic hydroxyl groups. This makes DNA acidic.

•     At high ionic concentration, cations shield the negative charge and stabilize the DNA double helix.

Ionic induction:

•    As DNA is acidic, it is negatively charged. During agarose gel electrophoresis it moves towards the anode (+).  Through this process, DNA can be separated according to their weight.

G:C bond:

•  G:C bonds are more stable as they form three H-bonds and more favorable stacking interactions. So, the higher the GC content, the more stable is the DNA molecule.

Enzymatic hydrolysis:

•    DNA can be hydrolyzed by enzymes called DNases. The enzyme that can hydrolyze DNA sequentially from ends is called exonuclease (Ex: Exonuclease I). The enzyme that can hydrolyze phosphodiester bonds in DNA internally is called endonuclease (Ex: DNase I).

Conformations of DNA

•      Several structural variants of DNA are possible:

–     B-DNA (most prevalent conformation in physiological conditions)

–     A-DNA

–     Z-DNA

Z-DNA:

•      Z-DNA is a left-handed double helical structure with a zig-zag pattern.

•      Biologically active but transient structure induced by biological activity.

•      The major and minor grooves show little difference in width.

•      Conditions for Z-DNA:

–     Alternating purine-pyrimidine

–     Negative DNA supercoiling

–     Low salt and cation concentration

–     Physiological temperature 37 °C and pH 7.3 – 7.4

•      Significance:

–     Provides torsional strain relief (supercoiling) while DNA transcription occurs.

–     Potentiality to form Z-DNA also correlates with regions of active transcription

A-DNA:

•      A-DNA is a right-handed double-helical structure fairly similar to B-DNA.

•      Biologically active but transient structure induced by biological activity.

•      The structure is shorter and more compact.

•      Major grooves are deeper and minor grooves shallower than B-DNA.

•      Conditions for Z-DNA:

–     Dehydrated samples of DNA (ex: samples for crystallographic studies)

–     Found in DNA-RNA hybrids and double-stranded RNA.

Geometry attributes	A-DNA	B-DNA	Z-DNA
Helix sense	Right-handed	Right-handed	Left-handed
Repeating units	1 base pair (bp)	1 bp	2 bp
Rotation/bp	+33.6°	+36°	-30°
Mean bp/turn	11	10	12
The inclination of bp to the axis	+19°	-1.2°	-9°
Rise/bp along the axis	2.3Å	3.4Å	3.8Å
Rise/turn of helix	24.6Å	34Å	45.6Å
Mean propeller twist	+18°	+16°	0°
Glycosyl angle	anti	anti	Pyrimidine: anti; Purine: syn
Sugar pucker	C3’ endo	C2’ endo	C: C2’ endo; G: C2’ exo
Helix diameter	26Å	20Å	18Å

anti and syn glycosyl angle

The bond joining the 1′-carbon of the deoxyribose sugar to the heterocyclic base is the N-glycosidic bond. Rotation about this bond gives rise to syn and anti-conformations. Rotation about this bond is restricted and the anti-conformation is generally favored, partly on steric grounds.

Propeller twist

•      The bases in a base pair are usually not coplanar; they instead are twisted about the hydrogen bonds that connect them, like the blades of a propeller.

•      The dihedral angle that defines the non-coplanarity is called the propellor twist angle.

Sugar pucker

• The pentose sugar ring in DNA is not planar. This non-planarity is called sugar puckering.

• Sugar puckering can influence both nucleotide incorporation and extension by polymerases.

•    RNA polymerases preferentially incorporate nucleotides having C3’-endo pucker while DNA polymerases prefer C2’-endo pucker.

•  If the puckered C atom is displaced towards the C5 atom, then that twisted form is called endo. If it is displaced away from the C5 atom, the form is called exo.

•    For example, if C3 is twisted towards the C5, it is called C3’-endo.