DNA Replication and Repair

LECTURE TITLE

  • DNA Replication and Repair
    Dr. Chiara De Santi
    Date: 7th October 2025
    Year 1 DEM Module FFP1

LECTURE LEARNING OUTCOMES

  • By the end of this lecture, you should be able to:
    • Describe the process of DNA replication: Initiation, Elongation, and Termination.
    • Explain the function of key enzymes involved in eukaryotic replication.
    • Outline the role of Telomerase.
    • Discuss DNA repair mechanisms.

DNA REPLICATION

  • Definition: DNA replication is a biological process by which a cell makes an exact copy of its DNA before cell division.
  • Key Features of DNA Replication:
    • It is complicated due to the involvement of many proteins and enzymes as chromosomes are structurally complex.
    • Occurs in the nucleus during the S phase of the cell cycle.
    • Strictly regulated.
    • It is semi-conservative: Each new DNA molecule consists of one original strand and one newly synthesized strand.
    • Visualization:
    • Parental DNA -> Daughter Strand -> Daughter Strand

OVERVIEW OF DNA REPLICATION

  • Steps:
    1. Initiation: Double-stranded DNA unwinds.
    2. Elongation: Complementary strands are generated by strict Watson-Crick base pairing (A-T and C-G).
    3. Termination: Removal of RNA primer and filling of gaps.

INITIATION

  • Definition: The separation of two complementary strands occurs at the ‘Origin of Replication’.
  • Characteristics:
    • Eukaryotic cells have multiple sites characterized by a consensus sequence (short AT-rich regions).
    • DNA helicase: Separates the DNA strands.
    • Single-Stranded Binding (SSB) proteins: Bind to the strands to prevent re-association.
    • Topoisomerases: Regulate the twisting of DNA and prevent super-coiling.
    • Two ‘replication forks’ move outward in opposite directions from the origin, preparing the strands for replication.

ELONGATION

  • Key Details (1):

    • Primer Generation: Primase generates the ‘primer’ from RNA instead of DNA.
    • DNA Polymerase: Reads the single-stranded DNA as a template from 3’ to 5’ and synthesizes new DNA from 5’ to 3’ by aligning and adding nucleotides according to the sequence specified by the template (using Watson-Crick base pairing).

  • Key Details (2):

    • Two strands are replicated simultaneously at the replication fork, but DNA polymerase synthesizes DNA only in the 5' to 3' direction, leading to:
    1. Leading strand: Synthesized continuously as it follows the replication fork (template read from 3’ to 5’).
    2. Lagging strand: Synthesized discontinuously in pieces (Okazaki fragments), described as semi-discontinuous replication.

SEMI-DISCONTINUOUS REPLICATION

  • Leading Strand: Continuous synthesis.
  • Lagging Strand: Synthesized in segments (Okazaki fragments) due to priming at regular intervals by Primase, leading to sections that are synthesized from 5’ to 3’ but interrupted.

DNA POLYMERASE – DETAILS (1)

  • Substrate Specificity:

    • Active site of DNA polymerase can bind all four deoxyribonucleoside triphosphates (dNTPs) with catalysis occurring only when the correct dNTP is bound.
    • Base pairing induces an enzyme conformational change, activating the enzyme.

  • Proof-Reading:

    • DNA polymerase possesses 3’ to 5’ exonuclease activity that enables error correction by removing mismatched nucleotides from the 3' end of the new strand.

DNA POLYMERASE – DETAILS (2)

  • Human Cell Enzymes: Key polymerases involved in eukaryotic replication include:
    • Pol α:
    • Initiates replication by forming a complex with primase to synthesize a short RNA primer (7-10 nt)+ 15 dNTPs.
    • Lacks exonuclease activity and hence proofreading ability; moderately processive.
    • Pol δ and Pol ε:
    • Additionally replicate DNA and do not associate with primase, highly processive (10-100 nt/sec), and possess 3' to 5' exonuclease activity.
    • Pol ε: Synthesizes the leading strand.
    • Pol δ: Synthesizes the lagging strand.