Chapter 5 - Nucleic Acid Structure, DNA Replication, and Chromosome Structure

Chapter 11 Outline

• Biochemical Identification of the Genetic Material
• Nucleic Acid Structure
• Overview of DNA Replication
• Molecular Mechanism of DNA Replication
• Molecular Structure of Eukaryotic Chromosomes

Genetic Material

• Functions as a blueprint for the construction of living organisms.
• Enables organisms to survive in their environments.
• Must meet criteria of: Information, Replication, Transmission, Variation

Identification of the Genetic Material

• In the late 1800s, scientists hypothesized a chemical substance within cells responsible for traits transmission.
• Researchers believed chromosomes were the hereditary material, observed doubling and dividing during cell division and containing DNA and protein.
• In the 1920s to 1940s, scientists anticipated proteins to be the genetic material.

Griffith's Bacterial Transformation

• In the late 1920s, Frederick Griffith studied Streptococcus pneumoniae strains.
• Smooth (S) strain: secretes a polysaccharide capsule, causing fatal infections in mice, pathogenic.
• Rough (R) strain: does not secrete a capsule, unable to cause infections in mice.
• Griffith discovered transformation, where non-virulent (R) bacteria were transformed into virulent (S) bacteria by a substance in heat-killed (S) bacteria.

Avery, MacLeod, and McCarty

• Followed up on Griffith's work in the 1940s
• Isolated DNA, RNA, and protein from the S type bacteria
• Only purified DNA converted type R bacteria to type S.
• DNA is the genetic material.

Levels of DNA Structure

• Nucleotides form a single strand of DNA.
• Two strands form a double helix.
• In living cells, DNA is combined with proteins to form chromatin, packaged into chromosomes.
• A genome is the complete complement of an organism's genetic material.

Nucleotides of DNA

• Three components: phosphate group, Deoxyribose, nitrogenous base
• Purines: Adenine (A), Guanine (G)
• Pyrimidines: Cytosine (C), Thymine (T)

Nucleotides of RNA

• Three components: phosphate group, Ribose, nitrogenous base
• Purines: Adenine (A), Guanine (G)
• Pyrimidines: Cytosine (C), Uracil (U)

Conventional Numbering System

• In the sugar ring, carbon atoms are numbered 1' to 5'.
• 1' carbon is to the right of the ring oxygen.
• Phosphate group is attached to 5' carbon.
• 3'-OH group is crucial for linking nucleotides.

A DNA Strand

• Nucleotides are linked by covalent phosphodiester bonds to form a strand (polymer)
• Strand has a specific directionality
• Sequence: 5' – TACG – 3'

DNA Structure

• In 1953, Watson, Crick, and Wilkins proposed DNA's double helix structure.
• Key contributions: Rosalind Franklin's X-ray diffraction results, Chargaff's base composition studies, Linus Pauling's methods
• Using ball-and-stick models
• DNA structure has been proposed by many scientists.

Rosalind Franklin's X-Ray Diffraction

• Atoms in a substance scatter X-rays.
• Repeating structures create diffraction patterns related to atomic arrangements.
• X-shaped diffraction is characteristic of a helix.

DNA Base Composition

• Erwin Chargaff analyzed DNA base composition from different species.
• Results show similar amounts of adenine (A) and thymine (T), and cytosine (C) and guanine (G).
• Chargaff's rule: A=T and C=G.

Watson & Crick

• Compiled existing knowledge (from colleagues' experimental approaches)
• Tested several DNA structure models
• Identified the correct model consistent with known findings.
• Published DNA structure in 1953.
• Awarded the Nobel Prize in 1962.

Structure of DNA Double Helix

• Two deoxyribonucleotide strands form a helical structure with a sugar-phosphate backbone on the outside and bases on the inside.
• The two strands are antiparallel (opposite directionality).
• Stabilized by hydrogen bonding between nitrogenous bases (A-T, G-C pairs)
• Base sequences of two strands are complementary (e.g., 5'-GCGGATTT-3' pairs with 3'-CGCCTAAA-5'

Three proposed mechanisms for DNA Replication

• Semiconservative: Each new DNA molecule includes one original and one newly synthesized strand.
• Conservative: One new molecule includes both original strands while the other molecule contains two newly synthesized strands.
• Dispersive: New DNA molecules have segments of original and newly synthesized DNA interspersed.

Experiments of Meselson & Stahl

• In 1958, Matthew Meselson and Franklin Stahl differentiated between the three replication models.
• Used isotopic labeling (N15 and N14) to distinguish between parental and new DNA strands.
• Used density gradient centrifugation to observe resulting DNA bands in each generation.
• Their results supported semiconservative replication.

Semiconservative DNA Replication

• During replication, parental strands separate and serve as templates for new strands.
• New nucleotides are added based on complementary base-pairing rules (AT/GC).
• Result is two identical double-helix DNA molecules each with one original and one new strand.

Bidirectional Replication

• DNA replication begins at an origin of replication.
• Base-pairing is disrupted allowing strands to unwind.
• Replication proceeds outward in both directions from the origin (bidirectional replication).

Single Origin of Replication (Bacteria)

• DNA replication starts from a single origin.
• Replication proceeds outward in both directions until the entire chromosome is replicated.

Multiple Origins of Replication (Eukaryotes)

• Replication begins at multiple origins (replication forks).
• Replication forks move outward in both directions until the entire chromosome is replicated.

Molecular Events of DNA Replication

• Origin of replication is bound by proteins that unwind DNA to create a replication bubble.
• Two DNA helicases move in opposite directions to separate the DNA strands, requiring ATP energy.
• A replication fork, in each direction, is created where the strands have been separated.
• Unwinding DNA creates tightened coils ahead of the replication fork. DNA topoisomerase alleviates that strain.

Molecular Events of DNA Replication (Cont.)

• Single-strand binding proteins prevent single strands from re-forming a double helix.
• This ensures the parental strands' nitrogenous bases remain exposed so DNA polymerase uses them as templates.

DNA Synthesis

• The enzyme DNA polymerase synthesizes DNA, using a DNA template strand.
• Slides along template DNA, covalently linking new nucleotides to the 3'-OH of the last nucleotide.
• Correct hydrogen bonding between incoming dNTPs and the template strand is required for accurate synthesis.
• Incorporating dNTPs breaks high-energy bonds, which is exergonic.

Primers

• DNA polymerase needs a primer to start DNA synthesis. This process occurs in a 5' to 3' direction.
• An enzyme called DNA primase makes an RNA primer.
• The RNA primer must be removed and replaced with DNA later.

Leading and Lagging Strands

• The replication fork moves unidirectionally. Newly synthesized strands will be made at the replication fork.
• The two template strands have opposite directions
• New daughter strands must be synthesized antiparallel to its template
• DNA polymerase works in the 5' to 3' direction only.
• Leads to different replication methods for each strand: Leading and Lagging strands.

Leading Strand Synthesis

• A single RNA primer is initially created at the origin.
• DNA polymerase extends the primer, moving in the same direction as replication fork.
• DNA synthesis occurs continuously from the primer, forming a single long molecule.

Lagging Strand Synthesis

• Requires multiple RNA primers near the replication fork.
• Short DNA segments are synthesized (Okazaki fragments) in the opposite direction of the replication fork.
• RNA primers between Okazaki fragments are later removed and joined.

Primer Removal and Replacement

• A specific DNA polymerase removes RNA primers and fills gaps with DNA.
• The enzyme DNA ligase connects the RNA primer-free DNA segments.

DNA Ligase

• Seals DNA fragments after RNA primers have been removed and replaced with DNA.
• Joins Okazaki fragments on the lagging strand and adjacent DNA strands from multiple origins.

Proteins involved in leading and lagging strand synthesis in E. coli

• Several proteins carry out different steps in DNA synthesis.
• DNA polymerase III, DNA primase, DNA polymerase I, and DNA ligase are essential for both leading and lagging strand synthesis.

DNA Replication is Very Accurate

• DNA polymerase has high fidelity due to correct hydrogen bonding.
• The polymerase's active site prefers correct matches over mismatches.
• Polynucleotide proofreading removes incorrectly paired bases
• Cells and additional repair enzymes are used to ensure DNA accuracy.

Telomeres

• Regions at ends of linear eukaryotic chromosomes.
• Consist of short repeated DNA sequences (e.g., TTAGGG repeats).
• Special telomeric proteins bind to these sequences, protecting chromosome ends from damage or fusion.

End Replication Problem

• Linear DNA molecules encounter problems at the ends.
• DNA polymerase can only work from 5' to 3' but cannot replicate the very end of the lagging strand.
• The result is progressive shortening of chromosomes in each DNA replication cycle

Telomerase

• Telomerase is an enzyme with a protein and RNA component.
• It attaches many new copies of the DNA repeat sequence to the ends of chromosomes to prevent chromosome shortening.
• It binds and synthesizes new telomeric DNA, then moves down and repeats this process to lengthen the 3' end.