DNA Replication

DNA Replication Overview

  • Key Quote: Watson and Crick (1953) noted the specific pairing of nitrogenous bases suggests a copying mechanism for genetic material.

Pattern of DNA Replication

  • Each strand of DNA serves as a template for synthesizing its complementary daughter strand.

  • Result in "new" DNA molecules that are half old and half new (known as the semiconservative model of DNA replication).

Characteristics of DNA Replication

  • Speed: DNA replication is rapid, processing 6 billion nucleotides in a few hours.

  • Accuracy: The error rate is highly precise, at 1 error per 10 billion nucleotides.

  • Enzymatic Requirement: Requires many enzymes and proteins to initiate and sustain the replication process.

Components of DNA Replication

1. Origin of Replication

  • Definition: The specific site in DNA where replication begins.

  • **Process:

    • Hydrogen bonds between base pairs are disrupted.

    • DNA denatures, resulting in the double strand opening up to create a replication bubble.

    • Results in two single DNA strands.

  • Types of Chromosomes:

    • In prokaryotes, there is usually one circular chromosome origin (sometimes referred to simply as 'ori').

    • In eukaryotes, there can be hundreds or thousands of origins on a linear chromosome.

2. Replication Fork

  • Definition: Y-shaped regions at either end of the replication bubble where DNA strands are being unwound and replicated.

3. Helicase

  • Function:

    • An enzyme that moves along the DNA strand, opening up the double helix at the origin of replication, creating two single strands that move away from each other.

    • Uses energy to disrupt the hydrogen bonds holding the strands together.

4. Single-Strand Binding Proteins (SSBPs)

  • Function:

    • Binds to single-stranded DNA to prevent rejoining of the strands after they have been separated by helicase.

    • Also blocks complementary base pairing hydrogen bonds from forming prematurely.

5. Topoisomerase

  • Function:

    • Enzyme that alleviates supercoiling tension introduced by helicase's unwinding of the DNA strands.

    • Cuts one single strand of DNA, allows it to untwist, and then seals the break to relieve tension.

6. DNA Primase

  • Function:

    • Enzyme that synthesizes a short RNA sequence (primer) that is complementary to a DNA sequence.

    • This primer is necessary for DNA Polymerase to begin synthesis of the daughter strands.

    • Works in a 5' to 3' direction and occasionally makes mistakes, noted during the process of DNA replication.

7. DNA Polymerase (DNAP)

  • Definition: Also known as DNA-dependent DNA polymerase; responsible for synthesizing new DNA strands.

  • Functions:

    • Must initiate replication at a double-stranded region.

    • Works by adding deoxyribonucleotides, which are the building blocks (substrates) made from dATP, dTTP, dCTP, and dGTP.

    • Moves in the 5' to 3' direction while reading templates in the 3' to 5' direction.

    • Has a built-in proofreading function to ensure fidelity during synthesis.

    • Generally larger and slower than DNA primase but more reliable with a lower error rate.

8. Leading Strand vs. Lagging Strand

  • Leading Strand:

    • Synthesized continuously in the direction of the replication fork by DNA Polymerase as it follows helicase.

    • Built in a 5' to 3' direction.

  • Lagging Strand:

    • Synthesized discontinuously in small segments known as Okazaki fragments.

    • DNA Polymerase moves away from the replication fork, working in 5' to 3' direction.

    • Requires the synthesis of multiple primers and the eventual joining of the fragments.

    • Primers on the lagging strand are replaced by DNA, which is done by a special repair enzyme DNA Polymerase, and are subsequently sealed together by the enzyme DNA ligase.

9. Proofreading DNA

  • DNA Polymerase possesses a mechanism known as "last-base" proofreading, which checks the last base added to ensure accuracy.

  • Additional repair enzymes act after replication to correct any remaining errors.

10. Telomeres

  • Function:

    • Found at the ends of eukaryotic chromosomes; composed of repetitive sequences that do not code for genes and are vital for protection.

    • These regions prevent the loss of important DNA sequences during replication, as linear chromosomes tend to shorten with each round of replication due to the inability to fully replicate the ends.

  • Associated Factor:

    • Telomerase is an enzyme that rebuilds telomeres, extending the length of these protective regions, especially in germ-line cells, stem cells, certain immune cells, and some cancer cells that demonstrate a form of immortality.

PCR (Polymerase Chain Reaction)

  • Components Required in PCR Tube:

    • DNA Polymerase

    • Deoxyribonucleoside triphosphates (dNTPs)

    • Double-stranded DNA (template)

    • Primers (short DNA sequences)

    • Buffer

    • Magnesium ions (Mg2+)

  • Process:

    • Heat tubes to 95°C to denature the DNA, separating the strands.

    • Cool the tubes to allow primers to attach to their complementary sequences on the DNA template.

    • Warm the tubes to enable DNA Polymerase activity for the synthesis of new DNA strands through amplification.