BIOL 4426: Cellular Physiology - Cell Nucleus and DNA Mechanisms

BIOL 4426: Cellular Physiology - Electronic Lecture Notes (Rafael Perez-Ballestero, 2022)
Part IV: The Cell Nucleus

Replication and Cell Cycle, Repair and Recombination

A. Replication

1. Characteristics of Chromosome Replication

  • Semiconservative Replication

    • Newly synthesized strands are paired with old template strands, meaning one original strand is conserved in each new DNA molecule.

    • Demonstrated by the Meselson-Stahl Experiment (1958), which provided critical evidence.

    • Used $E. coli$ bacteria grown in a medium containing a heavy nitrogen isotope (15N) to label their DNA, then transferred to a light nitrogen isotope (14N) medium.

    • After one round of replication, the bacterial DNA was a hybrid of intermediate density (half 15N, half 14N), detected by density gradient centrifugation. This result ruled out both conservative (where two new strands pair together) and dispersive (where DNA fragments are mixed) replication models.

  • Bidirectional Replication

    • Growth of replication bubbles that expand bi-directionally from an origin, resulting in two replication forks moving in opposite directions.

    • Demonstrated through electron microscopy observations of the replication of the SV40 virus, a small circular DNA virus.

  • Specific Locations for Replication Start

    • Begins at Origins of Replication, which are specific DNA sequences defining where replication starts.

    • Recognized by the Origin Recognition Complex (ORC), a multi-protein complex that marks these sites.

    • Autonomous Replicating Sequence (ARS), a well-studied type of origin sequence found in yeast and prokaryotic cells. Eukaryotic origins are generally more complex and less defined than in prokaryotes.

  • Number of Origins of Replication in Eukaryotic Cells

    • Prokaryotic cells usually possess a single, high-efficiency origin of replication for their circular genome, enabling rapid duplication.

    • Prokaryotic cells, like $E. coli$, can replicate their circular genome at approximately 1000 bp/sec in about 30 minutes due to their smaller genome size (e.g., 4.6 million bp).

    • Mammalian cells replicate linear chromosomes more slowly at about 100 bp/sec.

    • The large eukaryotic genome (\approx 3 billion bp for humans) is replicated in around 8 hours during the S phase, necessitating approximately 10,000-100,000 origins of replication to complete synthesis within a reasonable timeframe.

2. Replication Machinery and Mechanisms

  • General Components & Steps

    • The fundamental components and sequential steps involved in DNA replication are remarkably similar in both prokaryotic and eukaryotic cells, highlighting evolutionary conservation.

  • Origin Recognition Complex

    • Recognizes the origin of replication and facilitates the initial localized unwinding of the DNA double helix with the help of helicase, an enzyme that separates the two DNA strands using energy derived from ATP hydrolysis.

    • Single-stranded DNA binding proteins then bind cooperatively to the separated strands to stabilize unwound DNA, preventing re-annealing and protecting from nucleases:

      • Eukaryotic: Replication Protein A (RPA)

      • Prokaryotic: Single Stranded Binding Protein (SSB).

  • Primase

    • A specialized RNA polymerase (not a DNA polymerase) that synthesizes a small RNA primer ( typically 5-10 nucleotides long) complementary to the DNA template. This primer provides the free 3'-hydroxyl (3'OH) group necessary for DNA polymerase to initiate new DNA synthesis, as DNA polymerases cannot start a new strand de novo.

  • DNA Polymerases

    • Enzymes that synthesize new DNA strands by adding nucleotides one by one to the 3' end of a growing strand, using existing DNA as a template and obeying base-pairing rules.

    • Characteristics:

      • Cannot unwind DNA (requires helicase).

      • Cannot initiate synthesis without a pre-existing primer (provided by primase).

      • Synthesizes all new DNA strands exclusively in the 5' \text{ to } 3' direction.

      • Eukaryotic cells have 5 main nuclear polymerases (\alpha, \beta, \gamma, \delta, \epsilon) with alpha (\alpha), delta (\delta), and epsilon (\epsilon) key for nuclear DNA replication. Prokaryotic cells have 3 main polymerases (I, II, III), with DNA Pol III being the primary replicative enzyme.

  • Synthesis of Both Strands at Replication Forks

    • Due to the 5' \text{ to } 3' synthesis directionality of DNA polymerase and the anti-parallel nature of DNA strands, synthesis occurs differently for the leading and lagging strands at each replication fork.

  • Leading Strand Synthesis

    • This strand is synthesized continuously in the 5' \text{ to } 3' direction, moving towards the replication fork.

    • Requires only one RNA primer from primase to start.

    • Synthesized continuously by Pol-epsilon (\text{Pol-} \epsilon) in eukaryotes, whose processivity (ability to stay bound to the DNA template) is greatly enhanced by PCNA (Proliferating Cell Nuclear Antigen), which forms a ring-shaped sliding clamp around the DNA.

  • Lagging Strand Synthesis

    • This strand is synthesized discontinuously in the direction moving away from the replication fork (still in the 5' \text{ to } 3' overall direction for each fragment).

    • Requires multiple RNA primers (synthesized by primase) along the template strand.

    • Pol-alpha (\text{Pol-} \alpha) extends for short stretches from each primer, effectively synthesizing a DNA/RNA hybrid segment, which is then replaced by Pol-delta (\text{Pol-} \delta) with its associated PCNA sliding clamp for more extensive synthesis.

    • Synthesized as short segments called Okazaki fragments (typically 100-200 bp in eukaryotes, 1000-2000 bp in prokaryotes). These fragments necessitate eventual removal of RNA primers by RNase H and FEN1 (Flap Endonuclease 1) and subsequent joining of the DNA segments by DNA ligase, which forms the final phosphodiester bond.

  • Other Enzymes Involved in Replication

    • Topoisomerases

    • Relieve torsional stress (positive supercoiling, where DNA becomes excessively overwound ahead of the replication fork) that occurs during DNA unwinding by helicase. They do this by temporarily cutting DNA strands, allowing the strands to rotate and release tension, and then rejoining the strands.

    • Two main types:

      • Type I: Cuts one strand of the DNA double helix, allowing the intact strand to pass through the break, then reseals the cut.

      • Type II: Cuts both strands of the DNA double helix, allows another double helix segment to pass through the break, then reseals the cuts. DNA gyrase is a well-known prokaryotic Type II topoisomerase.

    • Telomerase and Telomeres

    • Eukaryotic linear chromosomes face a unique challenge in replicating their very ends completely, known as the "end replication problem," where primers are removed from the lagging strand's absolute end, leaving a gap.

    • Telomeres are specialized repeated nucleotide sequences (e.g., TTAGGG in humans) found at the ends of linear chromosomes. They act as protective caps, preventing the loss of vital genetic information during successive rounds of replication.

    • Telomeres are added by telomerase, a unique ribonucleoprotein enzyme complex that contains its own RNA template (e.g., 3'-AACCCCAAC-5') to synthesize new telomeric DNA. This enzyme effectively extends the parental lagging strand template.

    • Telomerase is highly active during early development (e.g., in embryonic cells and germline cells) to maintain telomere length. However, in most adult somatic cells, telomerase activity is significantly reduced or absent. Consequently, these cells lose a small portion of telomeric DNA at their edges across each division, leading to progressively shorter telomeres, which is linked to cellular senescence (aging) and a limit to cell division.

3. Replication and the Cell Cycle

  • Integration in Eukaryotic Cell Cycle

    • DNA replication is tightly integrated into the eukaryotic cell cycle, ensuring that DNA is duplicated accurately only once per cell division.

    • G1 phase: Cellular growth occurs, and the cell prepares for DNA replication, checking for DNA integrity and availability of resources.

    • S phase: The synthesis phase, where DNA replication occurs. This is typically one of the longest phases of the cell cycle, taking about 8 hours in mammalian cells. During S phase, all chromosomal DNA is duplicated.

    • G2 phase: The cell continues to grow and synthesizes proteins necessary for mitosis, and a final check for DNA integrity and complete replication is performed.

    • M phase: Mitosis and cytokinesis, where the cell divides into two daughter cells.

  • Regulation of Replication

    • Replication is exquisitely regulated through various cell cycle checkpoints that monitor the progress of DNA synthesis and chromosome segregation.

    • Cyclin Dependent Kinases (CDKs), in association with specific cyclins, are master regulators that control cell cycle transitions. Their activity fluctuates throughout the cell cycle.

    • Specific Cyclin-CDK complexes corresponding to each phase promote or inhibit progression through the cell cycle phases by phosphorylating target proteins. For instance, G1/S-CDK activity is crucial for initiating DNA replication. Checkpoints (like the S phase checkpoint) arrest the cell cycle if DNA damage or incomplete replication is detected, allowing time for repair.

B. DNA Repair

1. Replication Errors and DNA Damage

  • Fidelity of DNA Replication

    • DNA replication exhibits exceptionally high fidelity, with an initial error rate of approximately 1 in 10,000 nucleotides added. This is primarily due to the stringent base-pairing requirements and the proofreading activity inherent in DNA polymerases.

    • The 3' \text{ to } 5' exonuclease activity of DNA polymerases allows them to detect and remove incorrectly paired nucleotides immediately after incorporation. This proofreading capability reduces the error rate significantly to about 1 in 10 million to 1 in 1 billion base pairs, thus maintaining genomic stability.

  • Chemical Changes Leading to Errors

    • Spontaneous chemical changes can occur within DNA bases, such as tautomerism (rare isomeric forms of bases leading to mispairing) or deamination (e.g., cytosine converting to uracil), which can result in mismatched bases or point mutations during subsequent rounds of replication if not repaired.

  • Environmental Factors

    • DNA can be damaged by various exogenous physical factors (e.g., UV radiation causing thymine dimers, ionizing radiation causing double-strand breaks) and chemical agents (e.g., mutagens that alter base structure, carcinogens that promote cancer development), further emphasizing the constant threat to genome integrity.

  • Consequences of Errors in DNA

    • The accumulation of unrepaired errors or mutations in DNA can have severe consequences, including leading to genetic diseases, the development of cancer, various degenerative diseases, and ultimately, cell death, underscoring the critical necessity of robust DNA repair mechanisms.

2. DNA Repair Mechanisms

  • Mismatch Repair System

    • This system specifically fixes single base pair mismatches (e.g., A-C, G-T pairs) or small insertions/deletions that escape DNA polymerase proofreading during replication.

    • The process includes several steps: recognition of the mismatch by specialized proteins (e.g., MutS and MutL in bacteria, or their eukaryotic homologs), localization of the newly synthesized strand, removal of the incorrect base and a surrounding segment using exonucleases, replacement of the excised segment by DNA polymerase, and finally, ligation of the DNA backbone by DNA ligase. In bacteria, the new strand is identified by its lack of methylation compared to the parental strand; in eukaryotes, nicks in the newly synthesized strand serve as signals.

  • Excision Repair System

    • A broad category of repair pathways that fix damage affecting one or more nucleotides that distort the DNA helix.

    • This includes Nucleotide Excision Repair (NER), which repairs bulky lesions like thymine dimers (formed by UV radiation) and other helix-distorting damage, and Base Excision Repair (BER), which excises altered or damaged single bases (e.g., deaminated, alkylated bases) that do not distort the helix.

    • Both systems involve a multi-step method: recognition of the damaged DNA segment, removal of the damaged DNA (either a single base or a larger oligonucleotide segment), synthesis of a corrected strand using the intact complementary strand as a template by DNA polymerase, and final ligation by DNA ligase.

  • End-Joining Repair System (NHEJ)

    • Non-Homologous End-Joining (NHEJ) is a crucial pathway that repairs potentially lethal double-strand breaks (DSBs) in DNA. It involves directly ligating the broken DNA ends together without using a homologous template.

    • This process often results in small deletions or insertions (mutations) at the repair site due to the absence of a template strand and the potential loss of nucleotides during processing of the break ends, making it an error-prone repair mechanism.

    • NHEJ is particularly important in G1 phase when a sister chromatid for homologous recombination is not available. Homologous recombination (discussed below) is a more accurate method for DSB repair.

C. Recombination

1. Definition and Mechanisms

  • Homologous Recombination

    • A genetic recombination process involving the exchange of homologous DNA blocks between two similar or identical DNA molecules or chromosomal regions. It is utilized in both prokaryotic and eukaryotic cells for DNA repair and genetic diversity.

  • The Holliday Model

    • A widely accepted model (proposed by Robin Holliday) that describes the mechanism for DNA exchange, particularly relevant for meiotic recombination and some forms of repair.

    • It includes the following sequential steps:

      1. Single strand break in homologous DNA molecules.

      2. Strand invasion: A free single strand from one DNA molecule invades the homologous double helix of another (mediated by proteins like RecA in bacteria or Rad51 in eukaryotes).

      3. Strand migration (branch migration): The point of strand exchange (Holliday junction) moves along the DNA, leading to increased exchange of genetic material.

      4. Formation of a Holliday structure (also known as a cross-strand exchange), a four-stranded DNA intermediate.

      5. Resolution: The Holliday junction is cleaved by resolvases in one of two ways, resulting in either "patch" recombinant products (non-crossover) or "splice" recombinant products (crossover) where flanking markers are exchanged.

    • Key proteins involved include RecA (in bacteria) and Rad51 (in eukaryotes, crucial for DNA repair by homologous recombination and meiosis) for facilitating strand invasion, with other proteins mediating the formation and resolution of the Holliday junction.

  • Functions of Homologous Recombination

    • Serves as a high-fidelity DNA repair method, especially for double-strand breaks, by using an undamaged homologous chromosome or sister chromatid as a template.

    • Plays a crucial role in promoting genetic diversity during meiosis (prophase I), enhancing evolutionary adaptability by shuffling alleles between homologous chromosomes through crossing over.