DNA Packaging and Chromosomes Overview

DNA Packaging and Replication

• DNA is very thin (2.0 nm) and fragile, but needs to be protected and accessible within a cell's nucleus (5-10 μm in diameter).
• DNA packaging is essential for gene expression and cellular responsiveness.
• DNA must be compacted into a 3-D conformation aided by DNA-binding proteins (like histones).

Chromosomes

• Chromosomes consist of a single DNA molecule associated with proteins.
• A karyotype displays the number and types of chromosomes present in a cell.
• Chromosomes are comprised of chromatin, a complex of DNA and proteins.

Normal Human Chromosomes

• Normal human cells contain 23 pairs of homologous chromosomes.
• 22 pairs are autosomes (same in males and females).
• 1 pair are sex chromosomes (XX in females, XY in males; X is homologous, Y is much smaller with fewer genes).

Three-Dimensional Structure of DNA

• DNA must be packaged extensively to fit within a cell.
• This compaction is aided by DNA-binding proteins, primarily histones.
• DNA is wrapped around histones, forming nucleosomes (11 nm in diameter).
• Nucleosomes coil into a 30 nm fiber, and further compact into radial loops (300 nm in diameter).
• The final structure forms metaphase chromosomes (700 nm in diameter).

Chromatin

• Chromatin is a complex of DNA and proteins forming eukaryotic chromosomes.
• Two classes of chromatin proteins:
  • Histones (4 core histones: H2A, H2B, H3, H4, and other histones)
  • Non-histone proteins
    • Structural proteins associated with chromosomes
    • Involved in gene regulation

Histones

• Five classes of histones (H1, H2A, H2B, H3, H4)
• Rich in lysine and arginine amino acids.
• Histones are basic proteins.

Nucleosomes

• Nucleosomes are the fundamental structural units of chromatin.
• A nucleosome core particle consists of 146 base pairs (bp) of DNA wrapped around an octamer of histone proteins (2 H2A, 2 H2B, 2 H3, 2 H4).

Importance of DNA Packaging

• DNA packaging allows DNA to fit within the cell.
• Protecting the DNA from damage.
• Efficient DNA transmission during cell division.
• Facilitating gene expression.

DNA Replication

• Replication is the process of duplicating the entire genome prior to cell division.
• Biological significance:
  • Extreme accuracy is essential for preserving genome integrity across generations.
  • In eukaryotes, replication is restricted to the S phase of the cell cycle.
    • Slower replication rates result in higher fidelity.
• Prokaryotic DNA polymerase: 1000 bases per second
• Eukaryotic DNA polymerase: 50 bases per second

Basic Rules of Replication

• Semi-conservative: Each new DNA molecule is composed of one original strand and a newly synthesized strand.
• Starts at the origin: replication initiates at specific sites on the DNA.
• Synthesis in the 5' to 3' direction: DNA polymerases add nucleotides only to the 3' end of the growing strand.
• Semi-discontinuous: DNA synthesis is continuous on the leading strand and discontinuous on the lagging strand.
• RNA primers are needed: DNA polymerase cannot initiate synthesis; RNA primers provide a starting point.

DNA Replication Bubbles and Forks

• Replication bubbles eventually fuse to form newly replicated strands.
• Replication forks are Y-shaped regions where DNA is unwound and new strands grow.
• Linear eukaryotic replication proceeds from multiple origins and produces two linear DNA molecules.

Direction of Replication

• DNA polymerases always add nucleotides to the 3' end of a growing strand.
• Replication proceeds in the 5' to 3' direction.

Semi-discontinuous Replication

• Leading strand synthesizes continuously in the 5' to 3' direction.
• Lagging strand synthesizes discontinuously in the 5' to 3' direction via Okazaki fragments.

RNA Primers

• RNA primers are short RNA sequences needed to initiate DNA synthesis.
• RNA primers are synthesized by primase.
• Primers are removed and replaced by DNA polymerase.

DNA Polymerase

• DNA polymerase I: removes RNA primers and fills in gaps with DNA, particularly on the lagging strand.
• DNA polymerase III: elongates the new DNA strands by adding nucleotides.

Okazaki Fragments

• Short, discontinuous DNA fragments synthesized on the lagging strand.
• DNA ligase links adjacent Okazaki fragments.

DNA Ligase

• Joins DNA fragments, especially Okazaki fragments, together.

Core Proteins at the Replication Fork

• Topoisomerases: prevent supercoiling ahead of the replication fork.
• Helicases: separate the DNA strands.
• Primase: synthesize RNA primers.
• Single-strand binding proteins: stabilize the single-stranded DNA.
• DNA polymerase: synthesizes new DNA strands.
• DNA ligase: joins Okazaki fragments.

Mechanism of DNA Replication

• Initiation: Proteins bind to DNA, opening it up for complementary base pairing.
• Elongation: Proteins add nucleotides to the growing strand to form new DNA.
• Termination: Proteins release the replication complex.

When and Where Replication Occurs

• In eukaryotes, replication occurs during the S phase of interphase.
• DNA replication takes place within the nucleus of eukaryotic cells.