🧬Molecular Biology Unit 4 – DNA Replication and Repair

Key Concepts and Terminology

• DNA replication the process of creating two identical copies of DNA from one original DNA molecule
• Semi-conservative replication each strand of the original double helix acts as a template for the synthesis of a new complementary strand
• Origin of replication specific sequence in the DNA where replication begins
• Replication fork the Y-shaped region where the DNA strands are separated and new strands are synthesized
• Leading strand the strand of DNA that is synthesized continuously in the 5' to 3' direction
• Lagging strand the strand of DNA that is synthesized discontinuously in short fragments called Okazaki fragments
• DNA polymerase the enzyme responsible for catalyzing the synthesis of new DNA strands
• DNA ligase the enzyme that joins the Okazaki fragments on the lagging strand to create a continuous strand of DNA
• DNA repair mechanisms processes that detect and correct errors or damage in DNA to maintain genomic integrity

DNA Structure Recap

• DNA is a double helix structure composed of two antiparallel polynucleotide strands
• The strands are held together by hydrogen bonds between complementary base pairs adenine (A) with thymine (T) and guanine (G) with cytosine (C)
• The sugar-phosphate backbone provides structural support and consists of alternating deoxyribose sugars and phosphate groups
• The nitrogenous bases (A, T, G, and C) are attached to the sugar molecules and project inward from the backbone
• The directionality of DNA strands is determined by the 5' and 3' ends referring to the carbon atoms of the deoxyribose sugar
  • The 5' end has a phosphate group attached to the 5' carbon
  • The 3' end has a hydroxyl group attached to the 3' carbon
• The antiparallel nature of DNA means that one strand runs in the 5' to 3' direction while the complementary strand runs in the 3' to 5' direction

Stages of DNA Replication

• Initiation begins at the origin of replication where proteins bind and separate the DNA strands forming the replication bubble
  • Helicase unwinds and separates the DNA strands by breaking the hydrogen bonds between base pairs
  • Single-stranded binding proteins (SSBs) stabilize the single-stranded DNA and prevent the strands from reannealing
• Elongation involves the synthesis of new DNA strands complementary to the template strands
  • Primase synthesizes short RNA primers complementary to the template strand providing a starting point for DNA synthesis
  • DNA polymerase III extends the new DNA strand by adding nucleotides complementary to the template strand in the 5' to 3' direction
    • The leading strand is synthesized continuously
    • The lagging strand is synthesized discontinuously as Okazaki fragments
  • DNA polymerase I replaces the RNA primers with DNA nucleotides
  • DNA ligase joins the Okazaki fragments to create a continuous strand
• Termination occurs when the replication forks from opposite directions meet and the newly synthesized strands are separated from the template strands
  • Topoisomerases help relieve the tension and supercoiling caused by the unwinding of DNA during replication

Enzymes and Proteins Involved

• Helicase unwinds and separates the DNA strands by breaking the hydrogen bonds between base pairs
• Single-stranded binding proteins (SSBs) bind to and stabilize single-stranded DNA preventing the strands from reannealing
• Topoisomerases relieve the tension and supercoiling caused by the unwinding of DNA
  • Type I topoisomerases create single-strand breaks and pass one strand through the break before resealing it
  • Type II topoisomerases create double-strand breaks and pass a double-stranded segment through the break before resealing it
• Primase synthesizes short RNA primers complementary to the template strand providing a starting point for DNA synthesis
• DNA polymerase III the main enzyme responsible for DNA synthesis extending the new DNA strand by adding nucleotides complementary to the template strand
  • It has a high processivity meaning it can add many nucleotides before dissociating from the template
  • It also has proofreading activity allowing it to remove incorrectly incorporated nucleotides
• DNA polymerase I replaces the RNA primers with DNA nucleotides
• DNA ligase seals the nicks between Okazaki fragments creating a continuous strand of DNA

Replication Mechanisms

• Semi-conservative replication each strand of the original DNA molecule serves as a template for the synthesis of a new complementary strand
  • The original strands are separated and each acts as a template
  • New strands are synthesized complementary to the template strands
  • The result is two identical DNA molecules each containing one original strand and one newly synthesized strand
• Bidirectional replication DNA replication proceeds simultaneously in both directions from the origin of replication
  • Two replication forks are formed moving in opposite directions
  • This mechanism allows for faster replication of large DNA molecules
• Continuous synthesis on the leading strand the new DNA strand is synthesized continuously in the 5' to 3' direction
  • DNA polymerase III adds nucleotides to the growing strand without interruption
• Discontinuous synthesis on the lagging strand the new DNA strand is synthesized in short fragments (Okazaki fragments) in the 5' to 3' direction
  • DNA polymerase III synthesizes each Okazaki fragment starting from an RNA primer
  • The fragments are later joined by DNA ligase to create a continuous strand
• Proofreading and error correction DNA polymerases have proofreading activity that allows them to remove incorrectly incorporated nucleotides
  • The 3' to 5' exonuclease activity of DNA polymerase III can excise mismatched nucleotides
  • This mechanism helps maintain the accuracy of DNA replication

DNA Repair Processes

• Mismatch repair corrects errors that occur during DNA replication when incorrect nucleotides are incorporated
  • Mismatch repair enzymes recognize and excise the mismatched nucleotide
  • DNA polymerase fills in the gap with the correct nucleotide and DNA ligase seals the nick
• Base excision repair removes damaged or modified bases (oxidized, alkylated, or deaminated) from DNA
  • Glycosylases recognize and remove the damaged base creating an apurinic/apyrimidinic (AP) site
  • AP endonuclease cleaves the phosphodiester backbone at the AP site
  • DNA polymerase fills in the gap and DNA ligase seals the nick
• Nucleotide excision repair removes bulky DNA lesions (thymine dimers, pyrimidine dimers) caused by UV light or chemicals
  • Enzymes recognize the distortion in the DNA helix caused by the lesion
  • Endonucleases make incisions on both sides of the lesion and remove the damaged segment
  • DNA polymerase fills in the gap using the undamaged strand as a template and DNA ligase seals the nick
• Double-strand break repair fixes breaks in both strands of the DNA molecule caused by ionizing radiation or chemicals
  • Homologous recombination uses the sister chromatid as a template to repair the break accurately
  • Non-homologous end joining directly ligates the broken ends but is more error-prone

Common Errors and Mutations

• Point mutations changes in a single nucleotide resulting from substitution, insertion, or deletion
  • Substitution replaces one nucleotide with another (transition or transversion)
  • Insertion adds one or more extra nucleotides
  • Deletion removes one or more nucleotides
• Frameshift mutations insertions or deletions that alter the reading frame of the genetic code
  • Frameshift mutations can lead to completely different amino acid sequences or premature stop codons
• Chromosomal mutations large-scale changes in the structure or number of chromosomes
  • Deletions remove a portion of a chromosome
  • Duplications repeat a portion of a chromosome
  • Inversions reverse the orientation of a chromosomal segment
  • Translocations transfer a segment from one chromosome to another
• Spontaneous mutations occur naturally due to errors in DNA replication or repair
  • Tautomeric shifts in DNA bases can lead to mispairing during replication
  • Depurination loss of a purine base (A or G) creates an AP site that can lead to mutations if not repaired
• Induced mutations result from exposure to mutagens (chemicals, radiation, or viruses) that damage DNA or interfere with its replication
  • Alkylating agents (nitrosamines, mustard gas) add alkyl groups to DNA bases leading to mispairing
  • Intercalating agents (ethidium bromide, proflavine) insert between base pairs and distort the DNA helix
  • UV light causes the formation of pyrimidine dimers that can lead to mutations if not repaired

Real-World Applications and Research

• DNA sequencing technologies (Sanger sequencing, next-generation sequencing) rely on the principles of DNA replication to determine the nucleotide sequence of DNA
  • These technologies have revolutionized fields such as genomics, personalized medicine, and evolutionary biology
• Polymerase chain reaction (PCR) a technique that amplifies specific DNA sequences using DNA polymerase and primers
  • PCR has numerous applications in molecular biology, genetic testing, forensic science, and infectious disease diagnosis
• CRISPR-Cas9 a powerful gene-editing tool derived from bacterial adaptive immune systems
  • CRISPR-Cas9 uses guide RNA to target specific DNA sequences and the Cas9 endonuclease to create double-strand breaks
  • This technology has the potential to correct genetic disorders, create disease-resistant crops, and develop novel therapies
• DNA damage and repair in disease DNA damage and defects in repair mechanisms are associated with various diseases
  • Xeroderma pigmentosum a rare genetic disorder characterized by extreme sensitivity to UV light and increased risk of skin cancer due to defects in nucleotide excision repair
  • Hereditary nonpolyposis colorectal cancer (Lynch syndrome) caused by mutations in mismatch repair genes leading to increased risk of colorectal and other cancers
  • Fanconi anemia a genetic disorder caused by mutations in genes involved in DNA repair leading to bone marrow failure and increased risk of leukemia and solid tumors
• Cancer and genomic instability the accumulation of mutations and chromosomal abnormalities is a hallmark of cancer
  • Oncogenes (Ras, Myc) and tumor suppressor genes (p53, BRCA1/2) are often mutated in cancer cells
  • Defects in DNA repair pathways (mismatch repair, nucleotide excision repair, double-strand break repair) can lead to increased mutation rates and genomic instability in cancer