DNA Replication Notes

Discovery of DNA as the Molecule of Inheritance

Early Challenges

• In the early twentieth century, it was unclear what the molecule of inheritance was.
• Although Morgan's work (circa 1910) showed genes reside on chromosomes, the composition of chromosomes (DNA and protein) left ambiguity as to which component was the genetic material.

Experiments Highlighting DNA's Role

Griffith's Experiment (1928)

• Fred Griffith's experiment with mice and bacteria demonstrated the concept of transformation.
• Experiment:
  • Mice injected with pathogenic bacteria died.
  • Mice injected with non-pathogenic bacteria lived.
  • Mice injected with heat-killed pathogenic bacteria lived.
  • Mice injected with a mix of heat-killed pathogenic and living non-pathogenic bacteria died.
• Living pathogenic cells were extracted from the dead mice in the final step, suggesting genetic material had transferred from the heat-killed pathogenic strain to the living non-pathogenic strain.
• Transformation: Change in a cell's genotype and phenotype due to assimilating foreign DNA.

Bacteriophages (Phages)

• Viruses that infect bacteria, used in genetic research.
• Structure: Simple, consisting of DNA or RNA within a protective protein coat.
• Bacteriophages attach to bacterial cells and inject their contents.

Hershey and Chase Experiment (1952)

• Confirmed that DNA is the genetic material of a phage.
• Experiment:
  • Used E. coli and a phage that infects it.
  • Radioactively labeled sulfur in the phage protein and phosphorus in the phage DNA.
  • Mixed phage with bacteria, then used a blender to break apart cells.
  • Centrifuged the mixture to separate the pellet (cells) from the liquid (phage).
  • Results:
    • Radioactive sulfur was in the liquid, indicating the protein coat remained outside the cell.
    • Radioactive phosphorus was in the pellet, indicating the DNA entered the cell.
• Conclusion: DNA carries the genetic material of the phage and infects/transforms bacteria.

Chargaff's Rules (1950)

• Irwin Chargaff studied the composition of DNA.
• DNA is a polymer of nucleotides, each with a nitrogenous base, sugar, and phosphate group.
• Findings:
  • The base composition of DNA varies between species, making DNA a more credible candidate for genetic material.
  • In any species, the number of adenine (A) bases equals the number of thymine (T) bases, and the number of guanine (G) bases equals the number of cytosine (C) bases.
• These findings became known as Chargaff's rules.

Structure of DNA: Watson and Crick's Model

Using Existing Evidence

• Watson and Crick relied on previous work by Maurice Wilkins and Rosalind Franklin, who used X-ray crystallography to study DNA structure.
• X-ray crystallography involves firing X-rays at crystals of a molecule and recording the diffraction patterns.
• Franklin's images provided key information about DNA's structure.

Key Findings from X-ray Crystallography

• DNA is helical.
• The images provided information on the width of the helix and spacing of bases.
• DNA consists of two strands forming a double helix.

Model Building

• Watson and Crick built models based on X-ray data and known DNA chemistry.
• Franklin determined that sugar-phosphate backbones are on the outside, with nitrogenous bases inside.
• Watson and Crick proposed that the backbones are antiparallel (running in opposite directions).

Antiparallel Structure

• One strand runs 5' to 3' (top to bottom), while the other runs 5' to 3' (bottom to top).
• 5' and 3' refer to the carbon atoms on the sugar molecule.
• Nitrogenous bases are linked via hydrogen bonds.

Base Pairing

• Initial models of like-with-like pairing (A with A, G with G, etc.) were inconsistent with the uniform width of the helix seen in X-ray data.
• Pairing a purine with a pyrimidine resulted in a uniform width.
• Specific Pairing:
  • Adenine (A) pairs with Thymine (T).
  • Guanine (G) pairs with Cytosine (C).
• This specific pairing explains Chargaff's rules: A=T and G=C.

Publication and Recognition

• Watson and Crick published their model in 1953 in the journal Nature.
• They, along with Maurice Wilkins, were awarded the Nobel Prize in 1962. (Rosalind Franklin had died in 1958 and could not be awarded the prize.)

DNA Replication

Watson and Crick's Insight

• Watson and Crick suggested that specific base pairing implies a copying mechanism.
• Because the two strands are complementary, each can serve as a template for a new strand.
• The parental strands separate, and new copies are made alongside each parental strand.

Models of Replication

• Semiconservative Model: Each new DNA molecule consists of one original strand and one newly synthesized strand.
• Conservative Model: The parental strands reassociate after acting as templates for new strands, restoring the original double helix.
• Dispersive Model: Each strand of DNA contains a mixture of old and newly synthesized parts.

Meselson and Stahl Experiment (1958)

• Supported the semiconservative model.
• Used heavy (15N) and light (14N) isotopes of nitrogen to label old and new DNA strands.
• Experiment:
  • Cultured bacteria in 15N medium, then transferred them to 14N medium.
  • Extracted DNA and used density gradient centrifugation to separate DNA by density.
  • Results:
    • First replication produced a single band of hybrid (15N-14N) DNA, ruling out the conservative model.
    • Second replication produced two bands: one light (14N-14N) and one hybrid (15N-14N), ruling out the dispersive model.

Significance

• Provided strong evidence for the semiconservative model of DNA replication.
• Demonstrated that DNA separates, copies, and results in strands that are half old and half new.

The Process of DNA Replication

Overview

• DNA replication is fast and accurate but involves numerous enzymes and proteins.
• It begins at the origin of replication, where the two strands separate to form a replication bubble.
• In eukaryotes, there are hundreds or thousands of origins of replication on each chromosome.

Replication Forks

• Replication proceeds in both directions from each origin until the entire chromosome is copied.
• At each end of the replication bubble is a replication fork, the point where DNA strands are separating.

Key Enzymes and Proteins

• Helicase: Untwists the double helix at the replication fork.
• Single-Strand Binding Proteins: Stabilize single-stranded DNA to prevent re-pairing.
• Topoisomerase: Relieves overwinding of DNA ahead of the replication fork.
• Primase: Adds an RNA primer to provide a 3' end for DNA polymerase to start adding nucleotides.
• DNA Polymerase: Catalyzes the elongation of new DNA at the replication fork.
• DNA Ligase: Joins DNA fragments together.

DNA Polymerase

• DNA polymerase can only add nucleotides to the free 3' end of a growing strand, meaning new DNA is elongated in the 5' to 3' direction.
• In bacteria, DNA polymerase can add 500 nucleotides per second. In human cells, it's about 50 nucleotides per second.
• This speed contributes to the rapid division of bacteria.

Leading and Lagging Strands

• Due to the antiparallel structure of DNA, replication occurs differently on each strand.
• Leading Strand: Synthesized continuously in the 5' to 3' direction towards the replication fork.
• Lagging Strand: Synthesized discontinuously in the 5' to 3' direction, away from the replication fork, in segments called Okazaki fragments.
• Okazaki fragments are joined together by DNA ligase.

Steps in Lagging Strand Replication

• Primase makes an RNA primer.
• DNA polymerase adds nucleotides to create an Okazaki fragment.
• DNA polymerase I replaces the RNA primer with DNA.
• DNA ligase joins the fragments.

Proofreading and Repair

• DNA polymerase has a proofreading ability to check for and correct errors.
• Mismatch Repair: Enzymes remove and replace mismatched bases.
• Nucleotide Excision Repair: A nuclease cuts out damaged segments, DNA polymerase fills the gap, and DNA ligase seals it.
• These mechanisms help correct errors caused by replication mistakes or exposure to chemicals/physical agents (e.g., cigarette smoke, X-rays).

Mutations

• Despite proofreading mechanisms, errors can still occur.
• Sequence changes, called mutations, may become permanent and passed on to the next generation.
• Mutations are the source of genetic variation upon which natural selection operates.

Important Enzymes

• Helicase: Unwinds DNA at the replication fork by breaking hydrogen bonds.
• Primase: Creates RNA primers to initiate DNA synthesis.
• DNA Polymerase: Adds nucleotides to synthesize new DNA strands in the 5' to 3' direction and proofreads the new strand.
• Ligase: Joins Okazaki fragments on the lagging strand and seals gaps in DNA.
• Topoisomerase: Prevents supercoiling by relieving strain ahead of the replication fork.

Question

• A researcher observes that the lagging strand isn't working right due to something wrong with the DNA polymerase. Which enzyme is most likely malfunctioning? Primase
• If primase malfunctions, no RNA primer is made and DNA polymerase cannot start or continue to build the lagging strand.