🐇Honors Biology Unit 7 – DNA Structure and Replication

Key Concepts

• DNA (deoxyribonucleic acid) stores genetic information in living organisms
• DNA is composed of nucleotide monomers which consist of a sugar, phosphate, and nitrogenous base
• The four nitrogenous bases in DNA include adenine (A), thymine (T), guanine (G), and cytosine (C)
• Complementary base pairing occurs between A-T and G-C through hydrogen bonds
• DNA replication is the process of making an identical copy of the original DNA molecule
• Semi-conservative replication results in one original strand and one new strand in each daughter molecule
• DNA polymerase catalyzes the addition of nucleotides during replication and proofreads for errors
• Mutations can occur during replication if errors are not corrected leading to changes in the genetic code

DNA Structure

• DNA is a double helix structure resembling a twisted ladder
• The sugar-phosphate backbones form the sides of the ladder while the nitrogenous bases form the rungs
• Nucleotides are linked together by phosphodiester bonds between the sugar of one nucleotide and the phosphate of the next
• The 5' end of DNA has a phosphate group while the 3' end has a hydroxyl group
• Antiparallel orientation of the two strands means they run in opposite directions (5' to 3' and 3' to 5')
• Purines (A and G) always pair with pyrimidines (T and C) maintaining a constant width of the DNA molecule
• The double helix structure is stabilized by hydrogen bonds between complementary base pairs and base stacking interactions
  • A-T base pairs form two hydrogen bonds while G-C base pairs form three hydrogen bonds

DNA Replication Process

• DNA replication occurs during the S phase of the cell cycle before cell division
• Replication begins at specific sites called origins of replication with the unwinding of the double helix
• Helicase enzyme breaks the hydrogen bonds between complementary base pairs separating the two strands
• Single-stranded binding proteins (SSBs) stabilize the single-stranded DNA preventing it from reannealing
• DNA primase synthesizes short RNA primers complementary to the single-stranded DNA providing a starting point for DNA synthesis
• DNA polymerase III binds to the primer-template junction and catalyzes the addition of nucleotides in the 5' to 3' direction
  • Leading strand is synthesized continuously in the 5' to 3' direction
  • Lagging strand is synthesized discontinuously as Okazaki fragments in the 5' to 3' direction
• DNA ligase seals the nicks between Okazaki fragments on the lagging strand creating a continuous strand
• Telomerase adds repetitive DNA sequences to the ends of linear chromosomes (telomeres) preventing loss of genetic information during replication

Enzymes Involved

• Helicase unwinds the double helix and separates the two strands of DNA
• Single-stranded binding proteins (SSBs) bind to single-stranded DNA preventing reannealing and protecting it from degradation
• DNA primase synthesizes short RNA primers (8-12 nucleotides) complementary to the single-stranded DNA
• DNA polymerase III is the main enzyme responsible for DNA synthesis adding nucleotides to the growing strand
  • Catalyzes the formation of phosphodiester bonds between the 3' hydroxyl group of the growing strand and the 5' phosphate of the incoming nucleotide
  • Proofreads the newly synthesized strand for errors and corrects them using its 3' to 5' exonuclease activity
• DNA polymerase I replaces the RNA primers with DNA nucleotides and fills in gaps between Okazaki fragments
• DNA ligase seals the nicks between adjacent Okazaki fragments creating a continuous strand of DNA

Replication Errors and Repair

• Replication errors can occur due to the incorporation of incorrect nucleotides, slippage of the DNA polymerase, or damage to the DNA template
• Mismatch repair corrects errors that escape the proofreading activity of DNA polymerase
  • Methyl-directed mismatch repair system in E. coli recognizes and corrects mismatched base pairs
• Nucleotide excision repair removes damaged or modified nucleotides (thymine dimers caused by UV light) and replaces them with the correct nucleotides
• Base excision repair corrects small chemical modifications of bases (deamination, oxidation) by removing the damaged base and replacing it
• Double-strand break repair mechanisms (non-homologous end joining and homologous recombination) repair breaks in both strands of DNA
• Defects in DNA repair mechanisms can lead to increased mutation rates and genomic instability associated with cancer and other genetic disorders

Lab Techniques and Applications

• Polymerase chain reaction (PCR) amplifies specific DNA sequences using a heat-stable DNA polymerase (Taq polymerase) and specific primers
  • Used in DNA fingerprinting, genetic testing, and detection of infectious agents
• DNA sequencing determines the precise order of nucleotides in a DNA molecule
  • Sanger sequencing uses dideoxynucleotides (ddNTPs) to terminate DNA synthesis at specific positions
  • Next-generation sequencing (NGS) technologies enable high-throughput and parallel sequencing of millions of DNA fragments
• DNA cloning involves inserting a DNA fragment into a vector (plasmid or viral DNA) and introducing it into a host cell for replication
  • Recombinant DNA technology allows the production of proteins (insulin) and genetically modified organisms (GMOs)
• CRISPR-Cas9 is a genome editing tool that uses a guide RNA to target specific DNA sequences for cleavage by the Cas9 endonuclease
  • Enables precise modification of DNA sequences for research and therapeutic applications

Real-World Connections

• DNA fingerprinting uses PCR to amplify specific regions of DNA (short tandem repeats) for identification purposes in forensic investigations and paternity testing
• Genetic testing analyzes an individual's DNA to determine the presence of mutations associated with genetic disorders (sickle cell anemia, cystic fibrosis)
• Personalized medicine uses an individual's genetic information to tailor medical treatments and drug therapies based on their specific genetic profile
• Ancient DNA analysis involves extracting and sequencing DNA from ancient remains to study evolutionary relationships, population migrations, and extinct species
• Genetically modified organisms (GMOs) have been engineered to express desired traits (herbicide resistance in crops, increased nutritional value in golden rice)
• Gene therapy aims to treat genetic disorders by introducing a functional copy of a gene into the patient's cells to replace a defective gene
• DNA data storage exploits the high information density and stability of DNA to store digital data in the form of DNA sequences

Review Questions

1. What are the four nitrogenous bases found in DNA, and how do they pair with each other?
2. Describe the structure of a nucleotide and how nucleotides are linked together to form a DNA strand.
3. What is the role of complementary base pairing in the double helix structure of DNA?
4. Explain the process of semi-conservative DNA replication and the role of each enzyme involved.
5. How does DNA polymerase ensure the accuracy of DNA replication, and what happens when errors occur?
6. Distinguish between the leading and lagging strands during DNA replication and how they are synthesized.
7. What are some common types of DNA damage, and how are they repaired by the cell?
8. Describe the polymerase chain reaction (PCR) and its applications in DNA fingerprinting and genetic testing.
9. How has DNA sequencing technology advanced our understanding of genetics and its potential applications in personalized medicine?
10. Discuss the ethical considerations surrounding the use of genetic information and genome editing technologies like CRISPR-Cas9.