👨‍👩‍👦‍👦General Genetics Unit 7 – DNA Replication and Repair

Key Concepts and Terminology

• DNA (deoxyribonucleic acid) stores genetic information in living organisms
• Nucleotides consist of a sugar (deoxyribose), phosphate group, and nitrogenous base (adenine, thymine, guanine, or cytosine)
• Complementary base pairing (A-T and G-C) enables accurate replication and transcription
• Antiparallel orientation of DNA strands refers to the opposite 5' to 3' directionality of the two strands
• Semi-conservative replication produces two identical copies of DNA, each with one original and one newly synthesized strand
  • Demonstrated by the Meselson-Stahl experiment using heavy isotope labeling
• Replication fork forms as DNA unwinds and separates during replication
• Okazaki fragments are short DNA segments synthesized on the lagging strand during replication

DNA Structure and Function

• Double helix structure discovered by Watson and Crick in 1953
  • Two antiparallel polynucleotide strands coiled around a central axis
  • Stabilized by hydrogen bonds between complementary base pairs and base stacking interactions
• Genetic information encoded in the sequence of nucleotide bases (A, T, G, C)
• DNA packaging involves histones and chromatin to fit within the nucleus
  • Nucleosomes are the basic unit of chromatin, consisting of DNA wrapped around a histone octamer
• DNA serves as a template for RNA synthesis (transcription) and self-replication
• Mutations in DNA can lead to genetic variation and, in some cases, genetic disorders
  • Point mutations involve single nucleotide changes (substitutions, insertions, or deletions)
  • Chromosomal mutations affect larger segments of DNA (translocations, inversions, or duplications)

DNA Replication Process

• Occurs during the S phase of the cell cycle, before cell division
• Initiation begins at specific sites called origins of replication
  • Eukaryotes have multiple origins, while prokaryotes typically have a single origin
• DNA helicase unwinds and separates the double helix, forming the replication fork
• Single-stranded binding proteins (SSBs) stabilize the separated DNA strands
• Primase synthesizes short RNA primers to initiate DNA synthesis
• DNA polymerases extend the primers, adding nucleotides in the 5' to 3' direction
  • DNA polymerase III is the main replicative polymerase in prokaryotes
  • DNA polymerases α, δ, and ε are involved in eukaryotic replication
• Leading strand synthesized continuously in the 5' to 3' direction
• Lagging strand synthesized discontinuously as Okazaki fragments
  • Okazaki fragments are later joined by DNA ligase to form a continuous strand
• Telomeres are repetitive DNA sequences at the ends of linear eukaryotic chromosomes
  • Telomerase, an RNA-dependent DNA polymerase, maintains telomere length during replication

Enzymes and Proteins Involved

• DNA helicase unwinds the double helix and separates the DNA strands
  • Fueled by ATP hydrolysis
• Single-stranded binding proteins (SSBs) bind to and stabilize single-stranded DNA
• Topoisomerases relieve tension and supercoiling caused by DNA unwinding
  • Type I topoisomerases create single-strand breaks, while Type II topoisomerases create double-strand breaks
• Primase synthesizes short RNA primers (8-12 nucleotides) to initiate DNA synthesis
• DNA polymerases catalyze the addition of nucleotides to the growing DNA strand
  • Require a template strand and a primer to initiate synthesis
  • Proofread and correct errors during replication
• DNA ligase seals nicks between Okazaki fragments on the lagging strand
• Sliding clamp (β-clamp in prokaryotes, PCNA in eukaryotes) encircles DNA and enhances polymerase processivity
• Clamp loader (γ-complex in prokaryotes, RFC in eukaryotes) loads the sliding clamp onto DNA

Replication Errors and Mutations

• Replication errors can occur due to base misincorporation, slippage, or damage to the DNA template
  • DNA polymerases have proofreading activity to correct most errors during replication
• Spontaneous mutations arise from DNA damage caused by endogenous factors
  • Deamination converts cytosine to uracil and adenine to hypoxanthine
  • Depurination results in the loss of a purine base (adenine or guanine)
• Induced mutations result from exposure to exogenous mutagens
  • UV radiation causes pyrimidine dimers and 6-4 photoproducts
  • Chemical mutagens (alkylating agents, intercalating agents) modify DNA structure
• Translesion synthesis (TLS) polymerases bypass DNA lesions, but are error-prone
• Mismatch repair corrects base mismatches and small insertion/deletion loops
  • MutS, MutL, and MutH proteins involved in prokaryotic mismatch repair
  • MSH and MLH proteins involved in eukaryotic mismatch repair

DNA Repair Mechanisms

• Base excision repair (BER) corrects small, non-helix-distorting lesions
  • DNA glycosylases remove damaged bases, creating an apurinic/apyrimidinic (AP) site
  • AP endonuclease cleaves the phosphodiester backbone at the AP site
  • DNA polymerase and ligase fill the gap and seal the nick
• Nucleotide excision repair (NER) corrects bulky, helix-distorting lesions
  • Recognition of the lesion by XPC-RAD23B or transcription-coupled repair factors
  • Excision of the damaged strand by XPF-ERCC1 and XPG endonucleases
  • Gap filling by DNA polymerase and ligation by DNA ligase
• Double-strand break repair occurs through homologous recombination (HR) or non-homologous end joining (NHEJ)
  • HR uses the sister chromatid as a template for accurate repair
  • NHEJ directly ligates the broken ends, which can introduce errors
• Mismatch repair (MMR) corrects base mismatches and small insertion/deletion loops
  • Recognition of the mismatch by MutS (prokaryotes) or MSH (eukaryotes) proteins
  • Recruitment of MutL (prokaryotes) or MLH (eukaryotes) proteins to coordinate repair
  • Excision of the mismatched region and resynthesis by DNA polymerase and ligase

Clinical and Research Applications

• Mutations in DNA repair genes can lead to genetic instability and cancer predisposition
  • Xeroderma pigmentosum (XP) results from defects in nucleotide excision repair
  • Hereditary nonpolyposis colorectal cancer (HNPCC) is caused by mutations in mismatch repair genes
• Inhibition of DNA repair pathways can sensitize cancer cells to chemotherapy and radiation
  • PARP inhibitors target base excision repair and are used to treat BRCA-deficient cancers
• DNA damage response (DDR) pathways are potential targets for cancer therapy
  • ATM and ATR kinases are central regulators of the DDR and cell cycle checkpoints
• Studying DNA replication and repair in model organisms (Escherichia coli, Saccharomyces cerevisiae) provides insights into conserved mechanisms
• High-throughput sequencing technologies enable genome-wide analysis of replication origins, mutation patterns, and DNA damage
• CRISPR-Cas9 gene editing relies on precise DNA repair mechanisms to introduce targeted modifications

Review and Practice Questions

1. What are the four nitrogenous bases found in DNA, and which bases pair with each other?
2. Describe the semi-conservative model of DNA replication and how it was demonstrated experimentally.
3. What is the role of DNA helicase in the replication process, and how does it contribute to the formation of the replication fork?
4. Explain the difference between continuous and discontinuous DNA synthesis on the leading and lagging strands.
5. What are Okazaki fragments, and how are they processed to form a continuous lagging strand?
6. List three key enzymes involved in DNA replication and describe their specific functions.
7. Distinguish between spontaneous and induced mutations, providing examples of each.
8. How do DNA polymerases ensure the accuracy of DNA replication, and what happens when errors occur?
9. Compare and contrast the mechanisms of base excision repair and nucleotide excision repair.
10. Discuss the clinical implications of defects in DNA repair pathways, such as xeroderma pigmentosum and hereditary nonpolyposis colorectal cancer.