Topic

DNA Packaging and Manipulation

Nucleotide Composition

• Deoxyribonucleic acid (DNA) is made up of nucleotides.

• Each nucleotide has three components:

  1. Pentose sugar: deoxyribose

  2. Phosphate

  3. Nitrogenous base

The Nitrogenous Base

• Nitrogenous bases are categorized into two types:

  1. Purine:

  • Structure: Two ring structure.

  • Examples: Adenine (A) and Guanine (G).

  1. Pyrimidine:

  • Structure: One ring structure.

  • Examples: Cytosine (C) and Thymine (T).

DNA Strand Formation

• Adjacent nucleotides are connected by phosphodiester bonds.

• This bond is a covalent bond formed between the 5’ phosphate of one nucleotide and the 3’ hydroxyl group of the next, forming the phosphodiester backbone.

Polarity of Polynucleotide Strand

• The 5’ to 3’ polarity is determined by the position of the phosphodiester bonds between nucleotides.

DNA Structure

• DNA is composed of two polynucleotide strands.

• Covalent bonds connect adjacent nucleotides in the same strand, while hydrogen bonds connect nucleotides in different strands.

• DNA is a helical molecule, characterized by a constant diameter and a constant twist.

Hydrogen Bonding

• Hydrogen bonds occur specifically between nitrogenous bases, with:

  • Two bonds between adenine (A) and thymine (T).

  • Three bonds between guanine (G) and cytosine (C).

• A high quantity of these relatively weak hydrogen bonds contributes significantly to the structural stability of DNA.

Knowledge Check

• Given a scenario where DNA molecule A contains 25% cytosine while DNA molecule B contains 10% adenine, the question is: Which DNA molecule would be easier to separate into single polynucleotide strands?

Antiparallel Strands

• The two polynucleotide strands in DNA run antiparallel, meaning one strand runs 5’ to 3’ and the other runs 3’ to 5’.

• This arrangement allows for optimal hydrogen bonding between bases to occur.

DNA Organization

Definitions

• Genome: The complete set of genetic information found in an organism.

• Chromosome: A unit or molecule of DNA containing genetic information.

• Gene: A segment of a chromosome that encodes a functional product.

Importance of DNA Packaging

• DNA must be packaged and organized for several reasons:

  • Size: DNA is longer than the cell size.

  • Protection: To prevent DNA damage.

  • Regulation: To regulate gene expression.

Comparison of Prokaryotic and Eukaryotic DNA Organization

• 1 Mbp = One million base pairs

Plasmid

• Plasmid: Extrachromosomal DNA that is self-replicating and inherited.

• Found in bacteria, fungi, and some plants.

• Size: 2-100 kbp (kilobase pairs).

DNA Organization in Bacteria

• Bacterial DNA is not uniformly distributed; the chromosome is localized to the nucleoid.

Chromosome Condensation in Bacteria

• The bacterial chromosome is condensed through:

  • Looping: DNA binding proteins stabilize interactions between loops, bending DNA for compaction.

  • Supercoiling: The DNA double helix coils upon itself to facilitate spatial organization.

DNA Organization in Eukaryotes

Chromatin Structure

DNA + Histones

strings on a bead

• Chromatin: A complex of DNA and organizing proteins that appears as ‘beads on a string.’

• Each ‘bead’ is a nucleosome.

• This structure is dynamic and undergoes significant changes during transcription and DNA replication.

Nucleosome Composition

• Histones: The primary proteins associated with DNA; they are small and positively charged.

• A nucleosome contains:

  • Histone octamer: Composed of 2x (H2A, H2B, H3, H4).

  • 147bp DNA: The section of DNA wrapped around the histone proteins.

  • Linker DNA(50bp): The DNA segment between nucleosomes.

Histone Functionality

• Histone tails: Flexible extensions that protrude from the nucleosome, consisting of 19 to 39 amino acids.

• Functions of histone tails include:

  • Binding to negatively charged DNA segments.

  • Interaction with other nucleosomes to compact chromatin further.

Chromatin Conformations

• Chromatin exists in two main conformations:

  1. Open conformation:(beads on a string) Approximately 10 nm in diameter.

  2. Compact conformation: Approximately 30 nm in diameter, formed by condensing nucleosomes into a more compact structure.

DNA Packaging Steps in Eukaryotes

1. DNA wraps around the histone octamer to form a nucleosome.

2. Multiple nucleosomes form a 10 nm fiber (open conformation).

3. Nucleosomes condense further to create a 30 nm fiber (compact conformation).

4. The 30 nm fiber forms loops, continuing the compacting process.

5. Tight coiling of these condensed loops results in a characteristic chromosome configuration.

DNA Manipulation.

Molecular Cloning

• Clones are biological entities that are genetically identical, which can include whole animals, cells, or DNA sequences.

• Molecular Cloning: The process of making a recombinant DNA molecule that is replicated in a cell.

• recombinant DNA (rDNA): DNA created by joining 2 or more different DNA molecules together

• Plasmid Characteristics1

1. Oriign of replication

2. High copy # ( multiple copies of the same plasmid in a single cell)

3. selctable marker ( allow us to kill all bacteria that dont have the recombinant DNA)(antibiotic resistance)

4. Multiple cloning site

Steps of Molecular Cloning

1. Obtain the DNA (gene) of interest.

2. Insert the DNA into bacterial plasmid DNA.

3. Introduce the recombinant DNA into bacteria to enable replication.

Enzymes in Molecular Cloning

• Restriction endonucleases: nucleases: cut DNA. Endo: inside

  • Recognize specific DNA sequences, known as restriction sites, often palindromic in nature.

    • Restriction site

    • palindrome (same forwards and bakcwards)

  • Cleave the phosphodiester backbone of both DNA strands.

  • Staggered cuts produce sticky ends that facilitate the joining of DNA fragments.

Types of Restriction Endonucleases

• Different restriction endonucleases recognize different restriction sites and produce various sticky ends. For example:

  • EcoRI: Cuts, creating a 5’ overhang.

  • HindIII: Cuts, creating a 5’ overhang.

  • SacI: Cuts, creating a 3’ overhang.

Sticky End Annealing

• Complementary sticky ends will spontaneously anneal, allowing the fragments to bond and forming a stable structure, illustrating the base pairing ability.

DNA Ligase

• DNA Ligase: An enzyme that forms a phosphodiester bond between the 5’ phosphate and 3’ hydroxyl groups of adjacent DNA strands, sealing nicks in the DNA.

Steps of Using DNA Ligase

1. Digest DNA: Cut both the insert and the plasmid DNA with the same restriction enzyme.

2. Anneal DNA fragments: Mix the digested DNA fragments together.

3. Seal the nicks in the phosphodiester backbone using DNA ligase.

molecular scissors = restriction endonuclease

molecular glue = 1. base pairing b/w sticky ends

                            2. DNA ligase

CRISPR-Cas

• Genome editing: Making targeted changes to an organism's genome.

• CRISPR: A new genomic editing technology developed in the 2010s that is a simple and powerful tool for genome modification.

Components of the CRISPR-Cas System

1. Cas9: An endonuclease that does not require a restriction site for action. (endonuclease = cutsDNA inside)

2. Guide RNA: Binds to Cas9, directing it to the target DNA sequence for processing.

Steps in CRISPR-Cas

1. Cas9 binds to the guide RNA.

2. The guide RNA base pairs with the target DNA sequence.

3. Cas9 cuts both strands of DNA at the target site.

4. The resulting double-strand break is repaired through either non-homologous end joining (NHEJ) or homology-directed repair (HDR).

NHEJ = sticks ends bac together, sometimes inserts a nucleotide, sometimes removes. nucleotide.

HDR = find dna with similar sequence and use it to repair the double strand break.

Anti-Phage Defense Systems

DNA Manipulation Systems

• Enzymes/systems involved in DNA manipulation:

  • Restriction endonucleases (cut dna)

  • DNA ligase ( forms phosphodiester bond)

  • CRISPR-Cas (cuts DNA)

Anti-Phage Defense Systems

• Bacteria can be infected by viruses known as bacteriophages (phages).

• Bacterial defenses against phage infections include:

  1. Restriction endonucleases

  • Encoded from gene in bacterial chromosome

  • cuts phage DNA - No viral DNA replication

2. CRISPR-Cas

• components of CRISPR locus on bacterial chromosome

            1. CRISPR ARRAY (palendrome)

• Clustered Regularly Interspaced Short Palendromic Repeats

• Contains repeated DNA sequences seperated by spacers containing phage DNA

        2. Cas genes

• Encode proteins of the CRISPR-Cas system (e.g., Cas9)

• APPLICATIONS
  
  CRISPR technology can be utilized for gene editing, allowing scientists to modify DNA sequences in various organisms.

Restriction Endonucleases

• These enzymes are encoded by genes in the bacterial chromosome, specifically designed to target and cut phage DNA, which prevents viral DNA replication.

CRISPR-Cas Components

• Components of the CRISPR locus in bacteria include:

  1. CRISPR array: This consists of Clusters of Regularly Interspaced Short Palindromic Repeats that contain repeated DNA sequences interspersed with spacers that include phage DNA sequences.

  2. Cas genes: Encode the proteins that form the machinery of the CRISPR-Cas system (e.g., Cas9).

Steps in CRISPR-Cas Mechanism

1. Spacer acquisition: Short sequences of phage DNA are inserted into the CRISPR array to serve as spacers for recognition in future infections.

2. CRISPR RNA (crRNA) biogenesis: The CRISPR array is transcribed and then processed to form short crRNAs, combining parts of a repeat and a spacer.

3. Interference phase:

   • I. The crRNA binds to the Cas endonuclease.

   • II. The crRNA then base pairs with the target phage DNA.

   • III. The Cas endonuclease facilitates the cleavage of the phage DNA, preventing viral replication and effectively neutralizing the threat of the virus in the bacterial cell.