Chapter 16 Bio
DNA: The Blueprint of Life
DNA's Role: DNA, found in chromosomes, carries genetic information that determines traits like biochemistry, anatomy, and even some behaviors. It gets passed down from parents to offspring. Each gene is a unit of heredity, consisting of a specific DNA sequence.
Evidence for DNA as Genetic Material: The Hershey-Chase Experiment
Background: While chromosomes contain both DNA and protein, the case for protein as the genetic material initially seemed stronger. To investigate which molecule carried genetic information, Alfred Hershey and Martha Chase conducted experiments using bacteriophages, viruses that infect bacteria, specifically bacteriophage T2, which infects Escherichia coli (E. coli).
Experimental Design: Hershey and Chase knew that T2 phages were composed almost entirely of DNA and protein, and could reprogram host cells to produce more phages. They wanted to determine which component entered the bacterial cell during infection.
Radioactive Labeling: They used radioactive isotopes to label phage components: radioactive sulfur (35S) for protein and radioactive phosphorus (32P) for DNA. This choice of isotopes was based on the fact that protein contains sulfur but not phosphorus, while DNA contains phosphorus but not sulfur.
Infection and Separation: Separate batches of E. coli cells were infected with either protein-labeled or DNA-labeled phages. Shortly after infection, the mixtures were agitated in a blender to separate phage components outside the bacteria from the infected cells. The mixtures were then centrifuged, forcing the heavier bacteria to form a pellet at the bottom, while lighter phage components remained in the supernatant.
Results and Conclusion:
Radioactivity was found in the supernatant (liquid) for the protein-labeled phages, indicating that the protein did not enter the bacteria.
Radioactivity was found in the pellet (bacteria) for the DNA-labeled phages, demonstrating that the DNA had entered the bacterial cells.
Furthermore, bacteria infected with the DNA-labeled phages released new phages containing radioactive phosphorus, further confirming that the injected DNA played a role in producing new viral components.
These findings led Hershey and Chase to conclude that DNA, not protein, serves as the genetic material of phage T2. Their experiment provided strong evidence supporting DNA as the hereditary material.
DNA Structure: A Double Helix with Complementary Base Pairing
Double Helix: DNA is structured as a double helix - two antiparallel strands running in opposite directions. The structure resembles a twisted ladder, with the "side rails" formed by a sugar-phosphate backbone and the "rungs" consisting of pairs of nitrogenous bases.
Backbone: Each repeating unit in the backbone is a nucleotide composed of:
A deoxyribose sugar
A phosphate group
A nitrogenous base
Nucleotides are linked together by covalent bonds between the phosphate group of one nucleotide and the sugar of the next, forming a continuous chain.
Base Pairing: The nitrogenous bases in DNA pair specifically:
Adenine (A) pairs with Thymine (T) via two hydrogen bonds.
Guanine (G) pairs with Cytosine (C) with three hydrogen bonds.
Chargaff's Rules: These pairing rules, discovered by Erwin Chargaff, explained his observation that in any species, the amount of A is roughly equal to T, and G to C.
Uniform Diameter: Purines (A and G) have two organic rings, while pyrimidines (C and T) have one ring. Only the pairing of a purine with a pyrimidine results in a uniform diameter for the double helix.
Directionality: Each DNA strand has a 5' end (with a phosphate group) and a 3' end (with a hydroxyl group). The two strands run antiparallel, meaning their 5' to 3' directions are opposite. This antiparallel arrangement is crucial for DNA replication and other processes.
DNA Replication: The Basis of Inheritance
Semiconservative Replication: DNA replication is semiconservative, meaning that each new DNA molecule consists of one original (parental) strand and one newly synthesized strand.
Meselson-Stahl Experiment (1958): This experiment, using different isotopes of nitrogen, provided evidence for the semiconservative model.
Procedure:
E. coli was cultured for several generations in a medium containing a heavy isotope of nitrogen, 15N. This ensured that the bacteria's DNA incorporated the heavier isotope.
The bacteria were then transferred to a medium containing only 14N, a lighter isotope.
Samples were taken after the first and second DNA replications.
DNA from the samples was extracted and centrifuged to separate DNA molecules based on their densities.
Results and Interpretation:
After the first replication, the DNA had an intermediate density, indicating that it contained both 15N and 14N. This result ruled out the conservative model, which predicted two distinct bandsāone heavy and one light.
After the second replication, two bands appeared: one with intermediate density and one with light density. This pattern supported the semiconservative model, which predicted the formation of both hybrid (15N-14N) and light (14N-14N) DNA molecules.
The dispersive model, which predicted all DNA would have a mixed density, was ruled out.
Origins of Replication: DNA replication starts at specific locations called origins of replication. The origin is a short stretch of DNA with a specific sequence of nucleotides.
Prokaryotes: Bacterial chromosomes usually have a single origin of replication.
Eukaryotes: Eukaryotic chromosomes have multiple origins, allowing for faster replication of their much larger DNA molecules.
Steps of Replication:
Initiation:
Proteins that initiate DNA replication recognize and bind to the origin of replication. They separate the two strands of DNA, creating a replication "bubble". In bacteria, replication proceeds in both directions from the single origin until the entire circular chromosome is copied. In eukaryotes, multiple replication bubbles form and eventually fuse, speeding up the replication process.
Unwinding and Stabilization:
Helicases: Enzymes that untwist the double helix at the replication forks, separating the two parental strands.
Single-Strand Binding Proteins: Bind to the unpaired DNA strands, preventing them from re-pairing.
Topoisomerases: Relieve the strain ahead of the replication fork caused by unwinding, by breaking, swiveling, and rejoining DNA strands.
Primer Synthesis:
Primase: Synthesizes short RNA primers (5-10 nucleotides long) that are complementary to the DNA template strands. These primers provide a free 3' OH group, which is essential for DNA polymerase to begin adding DNA nucleotides.
Elongation:
DNA Polymerases: Enzymes that catalyze the synthesis of new DNA strands by adding nucleotides to the 3' end of a pre-existing chain (either an RNA primer or a DNA strand). Two main DNA polymerases involved in E. coli replication are DNA polymerase III (DNA pol III) and DNA polymerase I (DNA pol I).
Direction of Synthesis: DNA polymerases add nucleotides only to the free 3' end, meaning DNA synthesis always proceeds in the 5' to 3' direction. This directionality, coupled with the antiparallel arrangement of the DNA strands, results in different modes of synthesis for the two new strands.
Leading Strand: Synthesized continuously in the 5' to 3' direction towards the replication fork. Only one primer is needed for DNA pol III to synthesize the entire leading strand.
Lagging Strand: Synthesized discontinuously in the 5' to 3' direction away from the replication fork, in short segments called Okazaki fragments. Each Okazaki fragment needs its own primer.
Finishing Touches:
DNA Polymerase I: Removes the RNA primers from both the leading and lagging strands and replaces them with DNA nucleotides.
DNA Ligase: Joins the Okazaki fragments of the lagging strand into a continuous strand. It also joins the 3' end of the DNA that replaced the primer on the leading strand to the rest of the leading strand.
The Trombone Model of DNA Replication: The trombone model proposes a mechanism for how the DNA replication machinery operates.
Stationary Complex: Instead of DNA polymerase moving along the DNA, the DNA might move through a stationary replication complex, possibly anchored to the nuclear matrix in eukaryotes.
Looping of Lagging Strand: The lagging strand template loops back through the complex, allowing DNA pol III on both the leading and lagging strands to synthesize DNA simultaneously. This looping resembles the slide of a trombone, hence the name of the model.
Coordinated Synthesis: This model helps explain how the leading and lagging strands are synthesized concurrently at the same rate despite their different modes of synthesis.
Maintaining the Integrity of DNA: Proofreading and Repair
Proofreading:
DNA polymerases can proofread their work as they add nucleotides.
If an incorrect nucleotide is added, DNA polymerase can remove the incorrect nucleotide and replace it with the correct one. This proofreading significantly reduces errors during replication.
Mismatch Repair: Even with proofreading, some errors can escape detection by DNA polymerase. Mismatch repair involves a set of enzymes that:
Recognize mismatched nucleotides in newly synthesized DNA.
Remove the incorrect nucleotide.
Replace it with the correct nucleotide using the original (parental) strand as a template. Defects in mismatch repair systems can increase mutation rates and are associated with some cancers.
Nucleotide Excision Repair: This mechanism repairs DNA damage caused by various factors, including UV radiation.
Damage Recognition: Enzymes detect damaged DNA segments, such as thymine dimers formed by UV light. Thymine dimers distort the DNA helix and can disrupt replication and transcription.
Excision: A nuclease enzyme cuts the DNA strand on either side of the damaged segment, removing the damaged portion.
Repair Synthesis: DNA polymerase fills in the resulting gap using the undamaged strand as a template.
Ligation: DNA ligase seals the newly synthesized segment to the rest of the DNA strand. The importance of nucleotide excision repair is highlighted by the disorder xeroderma pigmentosum (XP), where defects in repair enzymes lead to hypersensitivity to sunlight and increased risk of skin cancer.
The Challenge of Replicating Ends: Telomeres
End Replication Problem:
DNA polymerase can only add nucleotides to an existing 3' end.
At the ends of linear eukaryotic chromosomes, the removal of the final RNA primer on the lagging strand leaves a gap that cannot be filled by DNA polymerase. This is because there is no 3' end available for nucleotide addition.
Consequently, the newly synthesized lagging strand ends up shorter than its template, leading to progressive chromosome shortening with each round of replication.
Telomeres: The Solution:
Structure and Function: Telomeres are repetitive, non-coding DNA sequences at the ends of eukaryotic chromosomes. They consist of multiple repetitions of a short nucleotide sequence (e.g., TTAGGG in humans). Telomeres have two main protective functions:
They prevent the staggered ends of daughter DNA molecules from activating the cell's DNA damage response systems, which could otherwise lead to cell cycle arrest or cell death.
They act as a buffer zone, delaying the erosion of genes located near the ends of chromosomes during repeated rounds of replication.
Telomere Shortening and Aging: Although telomeres provide some protection, they do shorten with each round of replication. This shortening is thought to contribute to the aging process in some tissues and possibly even the whole organism.
Telomerase: The Countermeasure: Telomerase is an enzyme that can lengthen telomeres, counteracting the shortening that occurs during DNA replication.
Mechanism: Telomerase carries its own RNA molecule that serves as a template for adding telomere repeats to the 3' end of the DNA strand.
Activity: Telomerase is active in germ cells, ensuring that chromosomes in gametes have full-length telomeres and preventing the loss of genetic information across generations. It's also active in some stem cells and cancer cells, but generally not in most somatic cells.
DNA Packaging: Chromatin Structure
Chromatin: In eukaryotic cells, DNA is associated with proteins to form a complex called chromatin. This organization allows the long DNA molecules to fit into the nucleus and plays a crucial role in regulating gene expression.
Nucleosomes: The Building Blocks:
The basic unit of chromatin organization is the nucleosome.
A nucleosome consists of a segment of DNA wrapped around a core of eight histone proteins.
Histones are positively charged proteins that bind tightly to the negatively charged DNA.
The "tails" of histone proteins protrude outward from the nucleosome and are involved in regulating gene expression.
Higher-Order Structure:
Nucleosomes are further organized into a 10-nanometer fiber, resembling "beads on a string," where nucleosomes are connected by linker DNA.
This fiber can be further folded and looped, forming more compact structures.
Looped domains of chromatin may be attached to the nuclear lamina (a network of protein filaments lining the inner nuclear membrane) and/or the nuclear matrix (a network of fibers within the nucleus). These attachments contribute to the spatial organization of chromosomes within the nucleus.
Euchromatin and Heterochromatin:
Euchromatin: Loosely packed chromatin. Its DNA is more accessible to transcription factors, the proteins involved in gene expression. Therefore, genes in euchromatin are generally active or potentially active.
Heterochromatin: Highly condensed chromatin. Its DNA is less accessible to transcription factors, resulting in gene silencing. Heterochromatin is found in regions like centromeres and telomeres, and its formation can be influenced by histone modifications.
Chromosome Condensation During Mitosis:
During interphase, chromatin is relatively uncondensed, allowing access for DNA replication and transcription.
As the cell prepares for mitosis, chromatin undergoes significant condensation, forming distinct chromosomes.
This condensation is essential for the proper segregation of chromosomes into daughter cells during cell division.
DNA Binding Proteins: Although not explicitly mentioned in the sources, many proteins bind to DNA and play crucial roles in DNA replication, repair, transcription, and other processes.
Examples: Histones (involved in chromatin structure), helicases (unwind DNA), single-strand binding proteins (stabilize single-stranded DNA), DNA polymerases (synthesize DNA), DNA ligase (joins DNA fragments), and transcription factors (regulate gene expression) are all examples of DNA binding proteins.