Replication

Nucleic Acid Structure & Replication

Overview of Organizations of Life in Eukaryotes

  • Nucleus: Described as a library where genetic information is stored.
  • Chromosomes: Analogous to bookshelves that organize and house genes.
  • Genes: Represented as books containing instructions encoded in DNA.
  • Cell Consistency: Most cells in an organism share the same genetic libraries and book collections.
  • Role of DNA: Contains essential information for cellular growth and function.

Nucleotide Structure

  • Nucleotides are the building blocks of nucleic acids, composed of the following:
    • Phosphates: Can be 1, 2, or 3.
    • Sugar: Either ribose (in RNA) or deoxyribose (in DNA).
    • Base: Can be either purine or pyrimidine.
    • Nucleoside: Formed by a sugar and a base.
    • Nucleotide: Formed by a nucleoside and one or more phosphates.

Structure of a Nucleotide

  • A nucleotide is composed of:
    • Pentose Sugar + Phosphate + Base.
  • Representation:
    • 5’ Carbon, 4’ Carbon, 3’ Carbon, 2’ Carbon, and 1’ Carbon of the sugar demonstrate the structural layout around the phosphate and nitrogenous base.

Chemical Properties of RNA and DNA

  • Ribose (RNA): Contains a hydroxyl group at the 2' position (2’OH), which gives RNA unique chemical properties.
  • Deoxyribose (DNA): Lacks the hydroxyl group at the same position, making DNA structurally distinct from RNA.

DNA Structure: A, B, and Z Forms

  • A-DNA:

    • Characteristics: Shorter due to a different sugar pucker; bases shifted away from the helical axis.
    • Structural results: Produces a cavernous major groove and a shallow minor groove; base pairs significantly tilted.

  • B-DNA: Standard form of DNA, typically the most studied morphology.

  • Z-DNA:

    • Occurs in G-C rich sequences and is characterized as a left-handed helix with a jagged backbone.
    • Requires high salinity to form and involves a sugar pucker shift from C2’ endo to C3’ endo for guanine nucleotides without changing the conformation for cytosine.

RNA Types in Humans

  • Major types include:
    • mRNA (messenger RNA)
    • rRNA (ribosomal RNA)
    • tRNA (transfer RNA)
  • Primary Structure Definition: Determined by the sequence of ribonucleotides.

Mature Eukaryotic mRNA Structure

  • Structure includes:
    • CAP Structure: Added post-transcription, not encoded within the gene.
    • Poly(A) Tail: A series of adenines added to the 3' end after transcription.
    • Start Codon: AUG and several stop codons represented.
    • Sequence Example: m7GpppN1 AAAAAAAAAA... with cap and poly(A) described.

tRNA Functionality

  • Functions as an adaptor molecule during translation.
  • Contains an anticodon that pairs with mRNA codons, e.g. UAG as shown in codon-anticodon pairing.
  • Crick's Adapter Hypothesis: Suggests that tRNA's role is matching amino acids to their corresponding codons on mRNA.

DNA Replication and Repair

  • Semiconservative Replication: Old strands serve as templates to synthesize new complementary strands.
Properties of DNA Polymerases
  1. Polymerization of deoxyribonucleotides occurs in the 5’ to 3’ direction.
  2. Templates are necessary for the replication process.
  3. Primers, typically short sequences of RNA, are required.

Biological Roles of DNA Polymerase I

  • Functions include:
    • Removing RNA Primers: Replaces RNA with DNA.
    • Filling Gaps: Performs DNA repair actions by synthesizing DNA to fill gaps resulting from RNA primer removal.
    • Processivity: Typically catalyzes around 20 nucleotide additions before dissociating from the template.
  • Enzymatic Activities:
    • 5’ → 3’ Polymerase: Adds nucleotides.
    • 3’ → 5’ Exonuclease: Allows proofreading of the recently added nucleotides.
    • 5’ → 3’ Exonuclease: Used to remove primers or erroneous bases.

Fidelity of DNA Replication

  • Operates under Watson-Crick geometry but has exceptions:
  • Tautomeric Forms: Occasional formation of non-Watson-Crick pairs (wobble pairs) lowers fidelity.
  • Error Frequency: Approximately $10^{-9}$, maintained by several safeguards:
    1. DNA polymerase has a binding pocket that prevents wobble pairs from facilitating catalysis.
    2. Tautomeric forms of bases (e.g., enol forms) are unstable.
    3. Editing activities of exonuclease enable removal of wrong bases by allowing DNA polymerases to “try again” after errors.
    4. Mismatch repair systems operate post-replication to correct any missed errors.

Editing Mechanism through Exonuclease Activity

  • The mismatch leads to initiation of a switch between polymerase and editing modes, particularly concerning the 3’ → 5’ exonuclease activity.

Directionality and Priming Problem

  • DNA can only be synthesized in the 5’ to 3’ direction, creating challenges during replication.
  • Semidiscontinuous Synthesis: Lead strand synthesized continuously, while the lagging strand synthesizes DNA in short Okazaki fragments.
  • Priming Problem: Due to the requirement for primers, RNA primers are synthesized to start the replication process.

The Bidirectionality Problem

  • Synthesis of DNA occurs bidirectionally, while maintaining semidiscontinuity on the lagging strand.
  • In bacteria, Okazaki fragments measure approximately 1000-2000 base pairs.

aA

E. Coli Replication

  1. Helicases: Unwind DNA utilizing ATP hydrolysis.
  2. Singe-Stranded Binding Proteins (SSB): Stabilize separated strands.
  3. Primosome: Responsible for synthesizing RNA primers for the lagging strand.
  4. DNA Polymerase III: Acts as the primary replicative enzyme.
  5. DNA Topoisomerase II: Relaxes supercoiled strands ahead of the fork.
  6. DNA Polymerase I: Replaces RNA with DNA.
  7. DNA Ligase: Joins Okazaki fragments together.

Processivity of DNA Polymerase III

  • Sliding clamps ensure the continual processivity of the DNA polymerase III during replication.

Summary of DNA Replication Paradigms

  • Semiconservative: Each DNA molecule splits to create two new strands.
  • Bidirectional: Replication occurs in two directions from a single origin.
  • Semidiscontinuous: Describes the differing continuity of leading and lagging strands.
  • RNA Primed: Primers are made of RNA to initiate replication.

Eukaryotic Replisome Components

  • Pol δ: Eukaryotic replicative polymerase.
  • Pol α/primase: Contains both primase and DNA polymerase activities.
  • PCNA: Trimeric clamp loader essential for stability during elongation.
  • Replication Factor C (RFC): Facilitates loading of the clamp.
  • MCMs: Helix unwinding complex composed of a hexa-meric formation.
  • RBPS (Replication Protein A): Stabilizes single-stranded DNA.
  • RNase H: Specific enzyme for removing RNA in RNA/DNA hybrids.

Chromosome End Replication

  • Challenges arise as the replication of chromosome ends can lead to loss of genetic material.
  • Priming by Pol δ and other factors like telomerase is critical to maintain chromosome integrity.

Telomerase and Cellular Aging

  • Most somatic cells show low telomerase activity linked to aging; however, it is present in stem and cancer cells, promoting cell immortality.
  • Dyskeratosis congenita and aplastic anemia associated with telomerase RNA mutations.
  • The link between telomerase activity and aging can manifest in cellular mechanics at the chromosomal level, contributing to lifespan variations.
  • Psychological stress correlates with reduced telomerase activity, accelerating telomere shortening, and mimicking a decade's worth of aging in studies of stressed caregivers.

Summary of DNA Replication

  • DNA replication involves:
    1. Identification of the origin of replication (OriC).
    2. Unwinding of double-stranded DNA to single-stranded.
    3. Formation of the replication fork.
    4. Synthesis of RNA primers by primase.
    5. Leading strand synthesis through DNA polymerase activity in the 5’-3’ direction.
    6. Lagging strand synthesis through Okazaki fragments.
    7. Removal of RNA primers and filling of gaps with dNTPs followed by ligation via DNA ligase.

Types of DNA Damage

  • Causes of DNA damage include:
    1. UV Damage.
    2. Environmental Chemicals: e.g., alkylating agents.
    3. Physiological Agents: hydrolytic deamination, depurination, oxidation.
    4. Replication Errors: mispaired bases.

Categories of DNA Damage Requiring Repair

  • Hydrolysis of glycosidic bonds (depurination).
  • Alkylation of bases (e.g., methylation of guanine).
  • Pyrimidine dimers from UV light.
  • Deamination (spontaneous or chemically induced).
  • Oxidative damage leading to strand breaks.

Induction of Pyrimidine Dimers

  • Occurs from UV exposure, causing adjacent thymines to form dimeric structures disrupting normal base pairing.

Hydrolytic & Oxidative Damage Analysis

  • Hydrolytic deamination catalyzed by nitrous acid and other agents lead to conversion of bases that can disrupt replication fidelity.
  • Rates of spontaneous deaminations:
    • A -> H: $10^{-9}/24$ hours.
    • G -> X: $10^{-9}/24$ hours.
    • C -> U: $10^{-7}/24$ hours (about 100 events/day).

Oxidative Damage Mechanisms

  • Reactive oxygen species (ROS) can cause extensive oxidative damage through various cellular processes, resulting in strand breaks and misincorporation during replication.

Consequences of 06-Methylguanine for Replication

  • Alters replication fidelity leading to mutations if not repaired.

Minimizing DNA Damage

  • Recommendations include avoiding harmful substances and environments:
    • Chemical exposure, processed foods, UV radiation, and oxidative stress.
  • Despite preventative measures, cells possess DNA repair pathways that correct damage after its occurrence.

DNA Repair Strategies

  • Include the following mechanisms and enzymes:
    • Base Excison Repair: Involves enzymes like Uracil-N Glycosylase and 8-oxoG Glycosylase.
    • Direct Reversal: Uses photolyase for dimers and MGMTase for alkylation damages.
    • Nucleotide Excision Repair: Different proteins operate in bacteria and eukaryotes.
    • Methyl-directed Mismatch Repair: Involves MutS, MutL, and MutH in bacterial cells and homologs in eukaryotes.

Mismatch Repair in Eukaryotes

  • Contains homologous systems to bacterial mismatch repair. Mutations in these systems can predispose to hereditary cancers such as hereditary nonpolyposis colorectal cancer (HNPCC).