Intercalating Agents and Supercoiling in DNA

Intercalating Agents

• Intercalating agents distort the double helix by inserting themselves between base pairs.
• Aromatic macrocycles are a type of intercalating agent.
• The binding of proteins to DNA is sequence-dependent, resulting in a gentle bending of the helix.

Superhelical Structure of DNA

• Supercoils are a form of tertiary DNA structure.
• Supercoils are introduced when a closed circle is twisted around its axis or when a linear piece of duplex DNA with fixed ends is twisted.
• Negative supercoils are produced when the molecule is twisted in the opposite direction of the clockwise turns of the right-handed double helix found in B-DNA.
• Positive supercoils overwind the DNA double helix.
• Negatively supercoiled DNA can arrange into a toroidal state, forming a more stable structure by wrapping around proteins.
• Supercoiled DNA is more compact and sediments faster during ultracentrifugation or migrates more rapidly in electrophoresis.

Prokaryotic DNA Supercoils

• DNA molecules in some organisms, such as bacteria, bacteriophages, and many DNA-containing animal viruses, as well as organelles like mitochondria, are circular with joined ends.

Eukaryotic Chromosomes

• Chromatin is the chromosomal material extracted from the nuclei of eukaryotic cells.
• Chromatin comprises very long double-stranded DNA molecules, a nearly equal mass of small basic proteins called histones, a smaller amount of nonhistone proteins, and a small quantity of RNA.
• The double-stranded DNA helix in each chromosome is thousands of times longer than the diameter of the cell nucleus.
• Histones condense the DNA.

Nucleosomes

• The basic unit of a eukaryotic chromosome is the nucleosome.
• Nucleosomes consist of DNA wound around a collection of histone molecules.
• The nucleosome contains approximately 200 base pairs of DNA.
• The main composition of the nucleosome includes the histone proteins H1, H2A, H2B, H3, and H4.

Histones

• Histones are a class of Arg- and Lys-rich basic proteins that interact with the anionic phosphate groups of the DNA backbone.
• The core histones undergo covalent modifications: acetylation, methylation, phosphorylation, ADP-ribosylation, monoubiquitylation, and sumoylation.

Nucleosome Structure

• 146 base pairs of DNA are wrapped around the surface of the histone octamer, forming the nucleosome core particle.
• The histone octamer consists of two copies each of histones H2A, H2B, H3, and H4.
• This structure protects the DNA from digestion by nucleases.

Higher-Order Structures of Chromatin

• The 10-nm fibril consists of 1.75 superhelical turns of DNA wrapped around the histone octamer, forming the nucleosome core particle.
• The core particles are separated by a 30-bp linker region of DNA.
• The 30-nm chromatin fiber is formed by the further supercoiling of the 10-nm fibril, with six or seven nucleosomes per turn.

Interphase Chromosomes

• Chromatin fibers in interphase chromosomes are organized into 30,000–100,000 bp loops or domains anchored to a scaffolding or supporting matrix within the nucleus.

Metaphase Chromosomes

• At metaphase, mammalian chromosomes exhibit twofold symmetry, with identical duplicated sister chromatids connected at a centromere, whose relative position is characteristic for a given chromosome.

Human Chromosomes

• Humans have 23 pairs of chromosomes.
• The human genome contains approximately 3 billion DNA subunits (bases: A, T, C, G).
• Approximately 23,000 genes code for proteins that perform most life functions.

Function of DNA

• DNA is the chemical basis of heredity, organized into genes, the fundamental units of genetic information.
• DNA provides a template for gene replication and transcription.
• Genes refer to specific fragments of DNA.
• Genetic information is stored in the nucleotide sequence of the gene.

Nucleic Acids

• The chemical bond linking nucleotides in the DNA molecule is a phosphodiester bond.
• The main point of the DNA double helix structure is the specific pairing of bases in the two strands.

RNA

• The sugar moiety in RNA is ribose, unlike the 2’-deoxyribose in DNA.
• RNA is susceptible to hydrolysis by base, whereas DNA is susceptible to hydrolysis by acid.
• RNA is a relatively labile molecule and undergoes easy and spontaneous degradation.
• RNA contains uracil instead of thymine.
• RNA exists as a single strand.
• The size of RNA is indicated by its sedimentation coefficient (S).
• RNA transcripts are complementary to the template strand of the gene from which they are transcribed.
• The guanine content of RNA does not necessarily equal its cytosine content, nor does its adenine content necessarily equal its uracil content.
• Many copies of RNA are present per cell.

Secondary Structure of RNA

• RNA molecules fold into stem-loop structures (hairpins) through intrastrand base pairing.
• Stems of RNA, paired regions forming a double helix conformation similar to A-form DNA, are the most prominent secondary structural elements.

mRNA Molecules

• The 3’-hydroxyl terminal of most mRNA molecules has an attached polymer of adenylate residues (20–250 nucleotides): the polyA tail.

5' Cap of mRNA

• The 5' cap of mRNA consists of 7-methylguanosine triphosphate linked to an adjacent 2'-O-methyl ribonucleoside at its 5'-hydroxyl through three phosphates.

Function of 5' Cap and PolyA Tail

• The 5' cap is involved in the recognition of mRNA by the translation machinery and helps stabilize the mRNA by preventing attack by 5'-exonucleases.
• The poly(A) "tail" maintains the intracellular stability of mRNA by preventing attack by 3'-exonucleases.

mRNA Function

• mRNA carries the nucleotide sequence as information from DNA and acts as a template for protein synthesis in ribosomes.

tRNA

• tRNA folds into a cloverleaf structure through intrastrand base pairing.
• tRNA molecules contain rare bases.

tRNA Arms

• tRNA contains five main arms or loops:
  • Acceptor Arm: At the 3' end.
  • Anticodon Arm: Contains the anticodon sequence.
  • DHU Arm: Contains dihydrouracil (DHU).
  • TΨC Arm: Contains pseudouridine and cytosine.
  • Extra (Variable) Arm: Varies in length.

Acceptor Arm

• The acceptor arm has the unpaired sequence cytosine, cytosine-adenine at the 3' end.
• These three nucleotides are added post-transcriptionally.

Nucleic Acid Hybridization

• Nucleic acid hybridization is a technique used to analyze nucleic acid structure and function.
• Hybridization is possible between DNA:DNA, RNA:RNA, and DNA:RNA.

Catalytic RNA (Ribozyme)

• Ribozymes are catalytic RNA molecules.
• They can act as sequence-specific endonucleases, degrading RNA.

Catalytic DNA (DNAzyme)

• DNAzymes are synthesized oligonucleotides that can degrade mRNA in a sequence-specific manner.

Denaturation of DNA

• DNA denaturation is the separation of the two strands of the double helix.
• Denaturation can be induced by acid, alkali, or heating.
• Hydrogen bonds between bases are disrupted during denaturation.
• The absorbance of denatured DNA at 260 nm is reduced.
• The melting temperature (Tm) of DNA is directly proportional to its GC content.
• Glycosidic bonds are not damaged during denaturation.

Tm Value

• Tm is the temperature at which 50% of the DNA double strands are opened.

Nucleic Acid Hybridization

• Single-stranded DNA from different samples can anneal and form a partial double helix in the presence of complementary base sequences.
• DNA can anneal with RNA to form a double helix.
• Nucleic acid hybridization can be used to study the structure and function of nucleic acids.
• It can identify sequence similarity between two nucleic acid molecules.