DNA Structure and Replication

DNA Structure and Replication

Chapter 13

Learning Goals

DNA

  • Apply Chargaff’s rule to predict base composition of a piece of DNA:
    Chargaff’s rules state that in any double-stranded DNA, the amount of adenine (A) equals the amount of thymine (T), and the amount of cytosine (C) equals the amount of guanine (G). This ratio can be used to determine the base composition of DNA samples.

Replication

  • Predict the relative melting temperature of a DNA helix based on its sequence:
    The melting temperature (Tm) of DNA is influenced by its GC content; higher GC content leads to a higher melting temperature due to the greater number of hydrogen bonds (three in GC pairs versus two in AT pairs).

  • Identify the complementary antiparallel strands making up a double helix:
    In a double helix, the two strands run in opposite directions (antiparallel), meaning one strand runs 5’ to 3’ while the other runs 3’ to 5’. Complementary bases (A-T and C-G) pair up across the strands.

  • Write down the complementary sequence of a given strand:
    Given a DNA strand’s sequence, the complementary sequence can be generated by replacing each base with its pair: A with T, T with A, C with G, and G with C.

  • Distinguish the 3’ vs. 5’ end of a DNA strand:
    The 5’ end of a DNA strand has a phosphate group attached to the 5th carbon of the sugar, while the 3’ end has a hydroxyl (-OH) group on the 3rd carbon.

  • Interpret Messelson and Stahl’s experimental results and distinguish between the three hypothetical models of DNA replication: Messelson and Stahl demonstrated that DNA replication was semi-conservative through experiments using isotopes of nitrogen to track DNA strands during replication. The three models of replication considered were:

    1. Conservative Model - parental strands remain together and the daughter strands are also associated together.

    2. Semi-conservative Model - each daughter DNA molecule consists of one parental and one new strand.

    3. Dispersive Model - parental and daughter DNA strands are intermixed.

  • List the reagents needed for a PCR reaction and describe what happens during each heat-cool cycle: Reagents needed for PCR include:

    • DNA template

    • DNA primers

    • DNA polymerase (thermostable)

    • Nucleotide triphosphates (dNTPs)
      Each cycle consists of three stages:

    1. Denaturation: Heating the mixture to around 94°C separates the DNA strands.

    2. Annealing: Cooling to around 50-65°C allows primers to bind to the template.

    3. Extension: Raising the temperature to around 72°C enables DNA polymerase to extend the primers, synthesizing new DNA strands.

  • Calculate the number of copies of a target sequence that will be produced by a given number of PCR cycles:
    The formula for calculating the number of copies produced after n cycles is:
    (2^n) where n is the number of cycles.
    For instance, after 3 cycles, there would be 8 copies produced.

  • Identify the roles of helicase, single-stranded binding proteins, sliding clamps, primase, DNA polymerase III, and DNA polymerase I:

    • Helicase: Unwinds the double helix.

    • Single-Stranded Binding Proteins (SSBPs): Prevent re-annealing of the unwound DNA strands.

    • Sliding Clamp: Stabilizes DNA polymerase on the DNA strand, increasing processivity.

    • Primase: Synthesizes a short RNA primer for DNA synthesis initiation.

    • DNA Polymerase III: Main enzyme that adds nucleotides to the growing DNA strand.

    • DNA Polymerase I: Removes RNA primers and replaces them with DNA.

  • Determine which direction a replication complex will move relative to a template strand:
    The replication complex moves along the template strand from the 3’ end to the 5’ end. Consequently, new strands are synthesized in the 5’ to 3’ direction.

  • Distinguish newly-built leading vs lagging strands:

    • Leading Strand: Synthesized continuously towards the replication fork.

    • Lagging Strand: Synthesized discontinuously away from the replication fork in fragments (Okazaki fragments).

  • Explain why and where Okazaki fragments are formed:
    Okazaki fragments are formed on the lagging strand due to the antiparallel nature of DNA strands; DNA polymerase can only synthesize in the 5’ to 3’ direction, which creates short segments when moving away from the replication fork.

  • Explain why the newly built lagging strand is shorter than the template strand, and how telomerase prevents this:
    The lagging strand is shorter because the RNA primers at each Okazaki fragment must be replaced with DNA, leaving the ends of the lagging strand unreplicated. Telomerase extends the telomeres, allowing for complete replication.

PCR and In Vivo DNA Replication are Slightly Different Processes

PCR

  • Replicates only the stretch of DNA between our chosen DNA primers.

  • Uses laboratory-synthesized DNA primers.

  • Denatures double-stranded DNA by heating the reagent mixture.

  • Requires a heat-tolerant DNA polymerase due to high temperatures involved.

In Vivo DNA Replication

  • Replicates the entire genome of the cell.

  • Utilizes RNA primers built by the enzyme primase.

  • Helicase and single-stranded binding proteins separate the double-stranded DNA, forming a replication bubble.

  • Contains multiple different DNA polymerases that perform various tasks during replication and are assisted by sliding clamp proteins.

Eukaryotes vs Prokaryotes

Replication Origins

  • Eukaryotes have multiple origins of replication (ori).

  • Prokaryotes, with their circular genome, have a single origin of replication (ori).

  • Components of a replication bubble:

    1. An ori sequence binds the pre-replication complex and initiates replication.

    2. A replication bubble consists of two replication forks that move away from each other during elongation.

Replication Direction

  • Replication proceeds in both directions from the origin of replication (ori).

    • As replication forks move away from each other during elongation, they ensure that the DNA strands are synthesized continuously.

Function of Primase

  • Primase adds new RNA bases complementary to the template strands:

    • Processive: An enzyme's ability to catalyze the same reaction many times without falling off.

    • DNA polymerase is indeed processive, as evidenced by its ability to remain attached to the DNA template during elongation.

Sliding Clamp

  • A sliding DNA clamp helps DNA polymerase work for more bases:

    • Without a sliding clamp protein, DNA polymerase III can add only dozens of bases before detaching from the template strand.

    • With a sliding clamp, it can add hundreds of bases, increasing efficiency of DNA synthesis.

Role of DNA Polymerase III

  • DNA polymerase III adds most of the new bases to the new strand:

    • It initiates synthesis at the RNA primer and continuously adds nucleotides complementary to the template strand.

Leading and Lagging Strand Synthesis

  • Which end of the new strand is DNA polymerase III adding bases to?

    • DNA polymerase III elongates both the leading and lagging strands.

    • DNA strands are created in the 5' to 3' direction: the template strand will have a 3' to 5' directionality.

    • The leading strand is synthesized continuously, while the lagging strand is synthesized in short segments (Okazaki fragments).

    • Single-stranded binding proteins hold the template strands apart during replication.

    • Primase synthesizes the RNA primer required for DNA polymerase to begin nucleotide addition.

Lagging Strand and Okazaki Fragments

  • Lagging Strand Process:

    1. Primase forms an RNA primer on the lagging strand template.

    2. DNA polymerase III adds nucleotides to this new Okazaki fragment at the 3' end until it hits the primer of the preceding fragment.

    3. DNA polymerase I hydrolyzes the RNA primer and replaces it with DNA.

    4. DNA ligase then catalyzes the formation of phosphodiester bonds, connecting these newly synthesized Okazaki fragments to complete the strand.

Okazaki Fragments

  • Okazaki fragments are generated along the lagging strand:

    • DNA replication can only occur in the 5’ to 3’ direction of the new strand.

    • Okazaki fragments produced on the lagging strand must be joined together by ligase after they are formed.

Mutations and Proofreading

  • Mutations can be caused during the cell cycle:

    • Errors in replication may occur by DNA polymerase during the synthesis process.

    • Most errors are repaired via the proofreading function of DNA polymerase, but some errors can become permanent mutations if not corrected.

  • Imperfect meiosis:

    • Problems such as nondisjunction and random breaking and rejoining of chromosomes can lead to mutations.

DNA Polymerase III Proofreading

  • Proofreading Function of DNA Polymerase III:

    1. During DNA replication, an incorrect nucleotide may be added to the growing DNA strand.

    2. Incidental errors in nucleotide addition will be recognized and excised by proteins of the replication complex.

    3. DNA polymerase III will then add the correct nucleotide to continue the replication process.

    • This proofreading ability is beneficial as it minimizes errors and enhances genetic fidelity during replication.

DNA Polymerase I and Repair Mechanisms

  • DNA Polymerase I can replace a damaged or incorrect base during DNA replication or at other times:

    • DNA Polymerase I carries out DNA repair either during replication or when damage is observed in the DNA strand at any time, thus maintaining genomic integrity.

Telomeres and Chromosomal Protection

  • Telomeres prevent chromosomes from being mistakenly joined together:

    • Telomeres consist of repetitive nucleotide sequences at the ends of chromosomes.

    • They bind protective proteins and form secondary structures to thwart unwanted cellular repair mechanisms from joining chromosomal ends together.

    • If the ends of varied chromosomes were to get joined, it could lead to genomic instability and functional failure.

End Replication Problem

  • Telomeres also protect against the end replication problem:

    • The lagging strand cannot be completely replicated due to the need for an RNA primer at its ends.

    • If coding DNA were situated at the ends of chromosomes, it could result in loss of genetic information during replication.

    • Telomeres extend beyond the coding regions, avoiding this loss.

Telomerase in Stem Cells

  • In stem cells, telomerase extends the telomere every time the DNA is replicated:

    • Presence of telomerase ensures no base pairs are lost during DNA replication, thereby maintaining chromosomal integrity.

    • Stem cells are equipped with telomerase allowing them to divide numerous times without losing essential genetic information.

Finite DNA Replication in Most Cells

  • DNA in most cells can only be replicated a finite number of times:

    • The requirement for RNA primers during replication limits the number of times DNA can undergo replication before critical segments of the genetic sequence are lost.

    • Telomerase acts to extend telomeres, counteracting this limitation and preventing loss of genetic sequences during replication cycles.

Summary

This document encompasses an exhaustive understanding of DNA structure, nucleotide pairing, replication mechanisms, and the critical roles of enzymes involved in the processes. Moreover, it emphasizes significant differences between PCR and in vivo DNA replication, highlights potential errors leading to mutations, and explains how telomeres and telomerase play crucial roles in maintaining genomic stability.