DNA Replication, Repair & Recombination – Comprehensive Page-by-Page Notes

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• Cells sustain internal order in a disordered universe by accurately duplicating DNA before cell division.

• DNA replication must precede formation of two genetically identical daughter cells.

• DNA is continually damaged by environmental chemicals, radiation, thermal accidents, and reactive molecules.

• Protein “machines” replicate and repair DNA with high speed and accuracy, illustrating cellular chemistry’s elegance.

• Short-term survival demands preventing DNA changes; long-term species survival requires some DNA variability.

• Occasional sequence changes (mutations) supply variation for natural selection.

• Chapter structure outlined: maintenance of DNA sequences, replication mechanisms, initiation/completion of replication, DNA repair, general recombination, site-specific recombination.

• Mutation = permanent change in DNA; can be lethal if in critical sequence positions.

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• Mutation rate (observable DNA changes) quantified in Escherichia coli.

  • E. coli divides ~30 min; large populations allow detection of rare mutants.

  • Example assay: lactose-utilization gene mutation detected using indicator dyes when cells grown on glucose.

• Many mutations are “silent” (synonymous codon change or conservative amino-acid change) → raw count underestimates true mutation rate.

• After correcting for silent mutations: average gene (~10^3 bp) mutates ~1 × 10⁻⁶ per cell generation.

• Therefore bacteria show ≈ 1 nucleotide change per $10^{9}$ nucleotides per generation.

• Germ-line mutation rate in mammals estimated indirectly:

  • Compare amino-acid divergence of same protein across species and fossil divergence time.

  • Use fixation rate of amino-acid change ≈ nucleotide alteration rate.

• Fibrinopeptides tolerate almost any amino-acid change (function discarded upon clotting), so accumulate mutations without selection pressure.

  • Data: typical 400-aa protein randomly altered once every ~2 × 10⁵ years in the germ line.

• DNA regions lacking critical information compared across species → mutation rate agrees with fibrinopeptide estimate.

• Normalized per round of DNA replication: bacteria and humans similar (~1 change per $10^{9}$ nt).

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• Plotting amino-acid differences vs divergence time gives straight line; slope = unit evolutionary time (time for 1 aa change/100 aa).

• Different proteins evolve at characteristic rates (Fig 5–1):

  • Histone H4 ≈ 1 change/100 aa in 500 My—highly constrained.

  • Cytochrome c ≈ 1/21 My.

  • Hemoglobin ≈ 1/5 My.

  • Fibrinopeptides very fast ≈ 1/0.7 My.

• Interpretation: fraction of random aa changes that are deleterious varies by protein.

• High deleterious mutation load imposes limit on acceptable mutation frequency:

  • With observed frequency, organism may manage ~60 000 essential genes.

  • Ten-fold higher frequency would cap complexity at ~6000 genes (evolution would stall < fruit fly).

• Somatic mutations create variant cells → natural selection inside body; extreme case cancer (~30 % mortality in EU & NA).

• Therefore high replication fidelity crucial both for germ-line stability and cancer prevention.

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• Summary mutation rate: ≈ $1 \text{ error} / 10^{9}$ replicated nucleotides → human genome (3 × 10⁹ bp) gains ≈3 mutations per cell division.

• Introduces DNA replication mechanisms; replication up to 1000 nt/s.

• DNA templating: complementary base-pairing (A–T, G–C) copies sequence after strand separation (Figure 5–2).

• DNA polymerase discovered 1957; substrates = dNTPs; requires single-stranded DNA template.

• Polymerization reaction: 3′-OH attacks α-phosphate of incoming dNTP, releases pyrophosphate (PP_i) (Figures 5–3, 5–4).

• Replication fork asymmetry: each old strand template for new strand → semiconservative replication (Figure 5–5).

• Early 1960s Y-shaped replicating structures detected on chromosomes (replication forks; Figure 5–6).

• Antiparallel helix means only 5′→3′ synthesis possible; no 3′→5′ DNA polymerase.

• Okazaki fragments discovered (1000–2000 nt in bacteria; 100–200 nt eukaryotes) synthesized 5′→3′ on lagging strand.

• Continuous “leading” strand vs discontinuous “lagging” strand (Figure 5–8).

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• DNA polymerase fidelity: ≈1 error/10⁵ during polymerization; total 1 / 10⁹ with proofreading + mismatch repair.

• Proofreading steps:

  1. Pre-insertion check: correct dNTP has higher affinity; conformational change before phosphodiester bond.

  2. Exonucleolytic proofreading: 3′→5′ exonuclease removes mispaired 3′ end; polymerase resumes (Figures 5–9, 5–10).

• 5′→3′ direction essential: if synthesis were 3′→5′, excision would remove activating triphosphate → chain termination (Figure 5–11).

• RNA primers synthesized by DNA primase initiate Okazaki fragments (~10 nt RNA every 100–200 nt eukaryotes) (Figure 5–12).

• RNA primer removal: RNAse H + DNA polymerase fill gap; DNA ligase seals nick (Figures 5–13, 5–14).

• Helicase (hexameric ring, ATP-hydrolysis) unwinds DNA up to 1000 bp/s (Figures 5–15, 5–16).

• Single-strand binding protein (SSB) prevents hairpins, stabilizes unwound DNA (Figures 5–17, 5–18).

• Sliding clamp (β-clamp in E. coli, PCNA in eukaryotes) loaded by clamp loader (ATP-dependent) holds polymerase on DNA (Figure 5–19).

• Fork coordination: leading and lagging polymerases form dimer; lagging strand forms loop (“trombone”) (Figures 5–20, 5–22).

• Strand-directed mismatch repair (MutS–MutL etc.) removes replication errors missed by polymerase proofreading (Figure 5–23).

• Topoisomerases resolve “winding problem”:

  • Topo I nicks one strand, swivels, reseals (Figure 5–25).

  • Topo II (gyrase in bacteria) makes double-strand break, passes duplex through, ATP-hydrolysis (Figures 5–26, 5–27).

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• Eukaryotic replication forks similar but slower (~50 nt/s) & smaller Okazaki fragments (100–200 nt).

• Additional complexity: nucleosomes.

• Eukaryotic lagging strand polymerases: Pol α/primase initiates, extends short DNA; Pol δ elongates with PCNA clamp (Figure 5–28).

• Replisome subunits summarized (Figure 5–21).

• Origins of replication:

  • Bacteria: single origin (oriC); initiator DnaA; methylation state regulates initiation (Figure 5–32).

  • Eukaryotes: multiple origins. Yeast autonomously replicating sequences (ARS) identified via plasmid assay (Figure 5–36); ORC binds; pre-RC with Cdc6 & Mcm helicase (Figure 5–38).

• Chromosome replication timing: clusters of origins (replicon families) activate sequentially during S-phase; euchromatin early, heterochromatin late (Figure 5–35).

• Coupling DNA synthesis with histone supply: histone genes amplified; histone mRNA ↑50× in S-phase; CAFs assemble nucleosomes behind fork; new H3/H4 acetylated then deacetylated (Figures 5–40, 5–41).

• Telomere replication: end-replication problem solved by telomerase (RNA template + reverse transcriptase) extends 3′ overhang → lagging strand filled (Figures 5–42, 5–43).

• Telomere structure: t-loop with shelterin proteins protects ends (Figure 5–44).

• Telomere length homeostasis (yeast feedback; Figure 5–45); telomerase off in most human somatic cells → replicative senescence; cancer cells often reactivate telomerase.

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• DNA damage types: oxidative, hydrolytic (depurination 5000 events/cell/day; deamination C→U 100/day), alkylation, UV-induced thymine dimers (Figures 5–46–5–48).

• If unrepaired, produce base substitutions or deletions during replication (Figure 5–49).

• DNA repair pathways:

  • Base Excision Repair (BER): DNA glycosylase flips out damaged base, AP endonuclease cuts backbone; DNA pol + ligase fill seal (Figure 5–50A & 5–51).

  • Nucleotide Excision Repair (NER): multi-enzyme complex excises 12–30 nt around bulky lesion (thymine dimer); repair synthesis + ligase (Figure 5–50B).

• DNA base chemistry aids detection (e.g., uracil not natural in DNA; 5-methyl-C deamination to T problematic; Figure 5–52).

• Double-strand break (DSB) repair:

  • Nonhomologous End Joining (NHEJ): Ku proteins, ligase; error-prone (Figure 5–53A).

  • Homologous Recombination (HR): uses sister chromatid; precise (Figure 5–53B).

• DNA damage induces SOS response in bacteria (RecA activation, error-prone polymerases) and checkpoints in eukaryotes (ATM kinase; diseases AT, XP; Table 5–2).

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• General (homologous) recombination generates genetic diversity and repairs DSBs.

• Characteristics: crossing-over, heteroduplex joint (Figures 5–54, 5–55).

• Meiotic recombination initiated by programmed DSBs; 5′ ends resected producing 3′ single-strand tails (Figure 5–56).

• DNA hybridization model for synapsis; annealing via random collisions (Figure 5–57) facilitated in vivo by RecA/Rad51 filaments on ssDNA (Figures 5–58, 5–59).

• Branch migration spontaneous vs RecA-directed (Figure 5–60); Holliday junction formation & isomerization (Figures 5–61–5–63).

• Resolution of Holliday junctions → crossover or non-crossover products (Figure 5–64).

• Gene conversion: mismatch repair within heteroduplex alters allele ratios (Figures 5–65, 5–66).

• Mismatch proofreading suppresses recombination between divergent sequences (Figure 5–68).

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• Site-specific recombination:

  • Transpositional (cut-and-paste, replicative) vs conservative.

• DNA-only transposons: transposase recognizes inverted repeats; excision & insertion; generates target site duplication (Figures 5–70–5–72).

• Retroviruses & retroviral-like retrotransposons: RNA → cDNA via reverse transcriptase; integrase inserts into genome (Figures 5–73–5–75).

• Non-retroviral retrotransposons (L1, Alu): target-primed reverse transcription; endonuclease nick yields 3′-OH primer (Figure 5–76).

• Mobile elements carry antibiotic-resistance genes in bacteria (Figure 5–69).

• Human genome: ~45 % derived from transposons; L1 active–mutation source; Alu expansion (Figures 5–77, 5–78).

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• Conservative site-specific recombination: recombinase recognizes two specific sites; depending on orientation yields integration, excision, inversion (Figure 5–79).

• λ-phage integrase inserts/excises viral genome at att sites via heteroduplex joint; reversible (Figures 5–80, 5–81).

• Biological uses: phase variation in Salmonella flagellin; bacteriophage lysogeny; engineered Cre-loxP systems for conditional gene activation/inactivation in mice or Drosophila (Figure 5–82).

• Overall, DNA replication, repair, recombination maintain genome integrity yet permit controlled variability essential for evolution.