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Chapter 6: DNA Replication and Repair

6.1 Overview of Variations in DNA

  • Differences in DNA produce variations among individuals of the same species.

  • Over evolutionary time, genetic changes lead to species distinctions (e.g., different hair colors in a family).

  • Variations in traits are underpinned by underlying genetic differences.

6.2 DNA Replication

6.2.1 Base Pairing and Template Function

  • DNA acts as a template for its own replication.

  • Base pairs: A pairs with T, G pairs with C.

  • Each strand has a primary (S strand) and a complementary strand (S' strand).

  • DNA sequences read from 5' to 3' direction; complementary strands read in opposite 5' to 3' direction.

6.2.2 Semiconservative Replication

  • DNA replication is semiconservative: each daughter DNA has one old strand and one newly synthesized strand.

  • DNA synthesis begins at the origin of replication, where initiator proteins unwind the DNA double helix.

6.2.3 Models of DNA Replication

  1. Semiconservative Model: Each new DNA molecule contains one old and one new strand.

  2. Dispersive Model: DNA molecules contain segments of both old and new DNA.

  3. Conservative Model: Original DNA strand remains intact while an entirely new strand is synthesized.

6.2.4 Experimental Evidence

  • Meselson and Stahl Experiments: Used cesium chloride gradient centrifugation to demonstrate semiconservative replication by showing intermediate density of the replicated molecules.

6.2.5 Replication Forks and Directionality

  • Two replication forks form at each origin and move in opposite directions.

  • Leading strand heads toward the replication fork while the lagging strand moves away, forming Okazaki fragments.

6.3 DNA Polymerase and Synthesis Direction

  • DNA polymerase synthesizes new strands in the 5' to 3' direction.

  • Incoming nucleoside triphosphates form base pairs with the template strands.

  • The replication fork is asymmetrical.

  • Lagging strand synthesized in short Okazaki fragments using a back stitching mechanism.

6.3.1 Proofreading by DNA Polymerase

  • DNA polymerase has proofreading ability, correcting errors by removing mispaired nucleotides as they are detected.

  • Different sites within the polymerase enzyme are dedicated to synthesis and proofreading functions.

6.3.2 Role of RNA Primase

  • RNA primers synthesized by RNA polymerase (primase) are necessary to start DNA replication.

  • Primase synthesizes RNA primers that are approximately 10 nucleotides long.

6.4 Enzymes and Proteins in DNA Replication

  • Essential enzymes include:

    • DNA Polymerases: Synthesize new strands and proofread.

    • RNA Primase (Primase): Synthesizes RNA primers.

    • DNA Ligase: Joins Okazaki fragments; functions like "glue".

    • Helicase: Unwinds the DNA double helix.

    • Topoisomerase: Relieves the torsional stress during unwinding.

    • Single-Strand Binding Proteins: Protect single-stranded DNA.

6.5 Telomerase and Chromosome Ends

  • Telomeres are GC-rich sequences at chromosome ends; telomerase extends these ends during DNA replication to prevent loss of sequences.

  • Critical for maintaining chromosome integrity during cell division.

6.6 DNA Repair Mechanisms

6.6.1 Types of DNA Damage

  • Depurination (loss of purine bases) and deamination (conversion of cytosine to uracil) lead to common DNA damages.

  • UV radiation can cause thymine dimers, linking adjacent thymine bases covalently.

6.6.2 DNA Repair Steps

  1. Excision: Damaged nucleotides are removed by nucleases.

  2. Resynthesis: DNA polymerase fills in the gaps using the complementary strand as a template.

  3. Ligation: DNA ligase seals any remaining breaks in the DNA backbone.

6.7 Mismatch Repair System

  • A repair mechanism that targets replication errors missed by proofreading.

  • Corrects single nucleotide mismatches that may result in permanent mutations if not repaired.

6.8 Double-Strand Break Repair

  • Double-strand breaks are repaired via:

    • Non-Homologous End Joining: Quick repair but may cause deletions.

    • Homologous Recombination: Preferred method that does not result in loss of nucleotides.

6.9 Consequences of DNA Repair Failures

  • Failure to repair can lead to mutations, potential diseases (e.g., sickle cell anemia), or genomic instability.

  • Importance of fidelity in DNA replication and repair is evidenced by comparisons of DNA sequences among organisms.