🧬Biochemistry Unit 7 – DNA Replication and Repair

Key Concepts and Terminology

• DNA replication process of duplicating DNA molecules during cell division
• Semi-conservative replication each newly synthesized DNA strand serves as a template for the next round of replication
• Origin of replication specific sites where DNA replication initiates
• Replication fork Y-shaped structure formed during DNA replication where the double helix is unwound
• Leading strand strand of DNA continuously synthesized in the 5' to 3' direction
• Lagging strand strand of DNA synthesized discontinuously in short fragments called Okazaki fragments
• DNA polymerases enzymes responsible for catalyzing the synthesis of new DNA strands
• DNA ligase enzyme that joins Okazaki fragments together to create a continuous strand of DNA
• Mutations changes in the DNA sequence that can lead to altered gene function or expression

DNA Structure and Function Review

• DNA composed of nucleotides consisting of a sugar (deoxyribose), phosphate group, and nitrogenous base (adenine, thymine, guanine, or cytosine)
• Double helix structure two complementary strands of DNA held together by hydrogen bonds between base pairs (A-T and G-C)
• Antiparallel nature DNA strands run in opposite directions with one strand oriented 5' to 3' and the other 3' to 5'
• DNA packaging DNA is tightly coiled around histone proteins to form nucleosomes, which further condense into chromatin fibers
• Genetic information DNA serves as a blueprint for the synthesis of RNA and proteins, which carry out cellular functions
• DNA stability double helix structure and base pairing contribute to the stability and integrity of the genetic material
• Central dogma of molecular biology flow of genetic information from DNA to RNA to proteins

Stages of DNA Replication

• Initiation replication begins at specific sites called origins of replication, where the double helix is unwound by helicase
• Primer synthesis short RNA primers are synthesized by primase to provide a starting point for DNA synthesis
• Elongation DNA polymerases extend the primers, adding nucleotides complementary to the template strand
  • Leading strand synthesized continuously in the 5' to 3' direction
  • Lagging strand synthesized discontinuously as Okazaki fragments
• Okazaki fragment maturation RNA primers are removed and replaced with DNA by DNA polymerase I, and the fragments are joined by DNA ligase
• Termination replication continues until the entire DNA molecule is duplicated, and the replication forks meet
• Proofreading and error correction DNA polymerases have proofreading activity to remove incorrectly incorporated nucleotides and maintain replication fidelity
• Telomere replication specialized mechanism to replicate the ends of linear chromosomes using telomerase

Enzymes and Proteins Involved in Replication

• Helicase unwinds the double helix by breaking the hydrogen bonds between base pairs
• Topoisomerases relieve the tension and supercoiling caused by the unwinding of the DNA helix
• Single-stranded DNA-binding proteins (SSBs) stabilize single-stranded DNA and prevent the formation of secondary structures
• Primase synthesizes short RNA primers complementary to the template strand
• DNA polymerases III (prokaryotes) and δ/ε (eukaryotes) synthesize the leading and lagging strands
  • Possess 5' to 3' polymerase activity and 3' to 5' exonuclease activity for proofreading
• DNA polymerase I (prokaryotes) and α (eukaryotes) replace RNA primers with DNA on the lagging strand
• DNA ligase seals the nicks between Okazaki fragments to create a continuous strand

Replication Mechanisms and Models

• Semi-conservative replication model proposed by Watson and Crick, each newly synthesized strand serves as a template for the next round of replication
• Replication bubbles multiple origins of replication in eukaryotic chromosomes lead to the formation of replication bubbles
• Bidirectional replication DNA synthesis proceeds in both directions from each origin of replication
• Replication fork progression coordinated action of enzymes and proteins at the replication fork ensures efficient and accurate DNA synthesis
• Okazaki fragment synthesis discontinuous synthesis of the lagging strand in short fragments of 100-200 nucleotides (prokaryotes) or 100-200 base pairs (eukaryotes)
• Sliding clamp (prokaryotes) and PCNA (eukaryotes) protein complexes that encircle the DNA and tether DNA polymerases to the template, increasing processivity
• Replication licensing system ensures that each origin of replication is used only once per cell cycle to prevent re-replication and maintain genome stability

DNA Repair Pathways

• Base excision repair (BER) repairs small, non-bulky lesions such as oxidized or deaminated bases
  • DNA glycosylases remove the damaged base, creating an apurinic/apyrimidinic (AP) site
  • AP endonuclease cleaves the phosphodiester backbone at the AP site
  • DNA polymerase fills the gap, and DNA ligase seals the nick
• Nucleotide excision repair (NER) repairs bulky lesions that distort the DNA helix, such as UV-induced pyrimidine dimers and chemically induced adducts
  • Damage recognition by XPC-RAD23B complex (global genome NER) or RNA polymerase stalling (transcription-coupled NER)
  • Excision of the damaged strand by endonucleases (XPF-ERCC1 and XPG)
  • Gap filling by DNA polymerase and ligation by DNA ligase
• Mismatch repair (MMR) corrects mismatched base pairs and small insertion/deletion loops that escape proofreading during replication
  • Recognition of mismatches by MutS homologs (MSH2-MSH6 or MSH2-MSH3)
  • Recruitment of MutL homologs (MLH1-PMS2) and endonuclease activity to create nicks flanking the mismatch
  • Excision of the mismatched strand, resynthesis by DNA polymerase, and ligation by DNA ligase
• Double-strand break repair repairs breaks in both strands of the DNA helix, which can be caused by ionizing radiation, chemicals, or replication fork collapse
  • Homologous recombination (HR) uses the sister chromatid as a template for accurate repair
  • Non-homologous end joining (NHEJ) directly ligates the broken ends, which can lead to small insertions or deletions

Mutations and Consequences

• Point mutations single nucleotide changes that can be substitutions (transitions or transversions), insertions, or deletions
  • Silent mutations do not change the amino acid sequence of the encoded protein
  • Missense mutations change the amino acid sequence and may affect protein function
  • Nonsense mutations introduce premature stop codons, leading to truncated proteins
• Frameshift mutations insertions or deletions that alter the reading frame, often resulting in nonfunctional proteins
• Chromosomal aberrations large-scale changes in chromosome structure or number (deletions, duplications, inversions, or translocations)
• Mutagenic agents external factors that increase the rate of mutations, such as UV radiation, ionizing radiation, and certain chemicals
• Mutator phenotype cells with defects in DNA repair or replication fidelity exhibit higher mutation rates
• Consequences of mutations can range from benign to deleterious, depending on the location and type of mutation
  • Loss-of-function mutations inactivate tumor suppressor genes, leading to uncontrolled cell growth and cancer
  • Gain-of-function mutations activate oncogenes, promoting cell proliferation and survival

Clinical and Research Applications

• Genetic disorders many inherited diseases are caused by mutations in specific genes (sickle cell anemia, cystic fibrosis, Huntington's disease)
• Cancer genomics identifying driver mutations and altered pathways in cancer cells to develop targeted therapies and biomarkers
• Personalized medicine using an individual's genetic information to tailor treatment strategies and predict disease risk
• Gene therapy introducing functional copies of genes into cells to correct genetic defects or treat diseases
• CRISPR-Cas9 gene editing a powerful tool for precise genome modification, with applications in basic research, biotechnology, and potential therapeutic use
• DNA forensics using DNA profiling to identify individuals based on unique genetic markers, with applications in criminal investigations and paternity testing
• Ancient DNA analysis studying the genetic material of extinct species or ancient human populations to understand evolutionary relationships and population histories
• Synthetic biology designing and constructing artificial biological systems or organisms with novel functions, such as producing pharmaceuticals or biofuels