Discuss and interpret the results of experiments that identified DNA as the genetic material
Frederick Griffith’s Transformation Experiment (1928): Griffith wasn’t trying to identify the genetic material, but rather, trying to develop a vaccine against pneumonia. He worked with two strains of Streptococcus pneumoniae bacteria: the R strain (nonvirulent) and the S strain (virulent). He found that heat-killed S strain could transform live R strain into virulent form. This suggested that some “transforming principle” from the S strain was taken up by the R strain, changing its characteristics
Avery, MacLeod, and McCarty’s Experiment (1944): These scientists identified the “transforming principle” in Griffith’s experiment as DNA. They treated heat-killed S strain with protein-digesting enzymes and injected it into mice along with live R strain. The mice still died, suggesting proteins were not the transforming principle. But when they treated heat-killed S strain with DNA-digesting enzymes, the mice survived. This suggested that DNA was the transforming principle
Hershey-Chase Experiment (1952): Alfred Hershey and Martha Chase provided further evidence that DNA is the genetic material. They used bacteriophages (viruses that infect bacteria), which are made of protein and DNA. They made two types of phages: one with radioactive sulfur to label proteins and one with radioactive phosphorus to label DNA. After infection, they found that the radioactive phosphorus (from DNA) was inside the bacteria, while most of the radioactive sulfur (from proteins) was outside. This indicated that DNA, not protein, was the genetic material
Describe the structure of nucleotides, a DNA strand, and the DNA double helix
- Nucleotides: a nucleotide is the base unit of DNA and it’s composed of three parts - a sugar called deoxyribose, a phosphate group, and one of four nitrogenous bases: Adenine, Thymine, Guanine, or Cytosine
- DNA stand: A polymer made of many nucleotides linked together. The sugar of one nucleotide bonds with the phosphate group of the next nucleotide, creating a sugar-phosphate backbone. The nitrogenous bases stick out from this backbone
- DNA Double Helix: Two DNA strands come together to form a double helix. The strands run in opposite directions, which is referred to as antiparallel. The nitrogenous bases from one strand interact with the bases from the other strand, forming base pairs. A with T and G with C through hydrogen bonds. This base pairing is complementary, and the strands twist together to form the double helix
Compare and contrast the structure of DNA and RNA
- Sugar: DNA contains the sugar deoxyribose, while RNA contains the sugar ribose. The difference is that ribose has one more hydroxyl group than deoxyribose
- Bases: DNA uses the bases adenine (A), thymine (T), guanine (G), and cytosine. RNA uses the bases adenine (A), uracil (U), guanine (G), and cytosine. So, in RNA, uracil replaces thymine
- Structure: DNA is usually a double-stranded molecule that forms a double helix, while RNA is typically a single-stranded molecule. However, RNA can sometimes form a secondary double helix structure.
- Function: DNA stores and transfers genetic information, while RNA converts the genetic information contained within DNA to a format used to build proteins, and then moves it to ribosomal protein factories
- Stability: RNA is less stable than DNA and is more vulnerable to mutation and attack
- Location: DNA is found in the nucleus, with a small amount of DNA also present in mitochondria. RNA molecules are made in the nucleus and can function in the cytoplasm
Despite these differences, DNA and RNA also share some similarities: they both store genetic information, are made up of nucleotides, and have a sugar-phosphate backbone
Discuss and interpret the work of Franklin, Chargaff, and Watson and Crick
Erwin Chargaff: Chargaff discovered two key rules that helped lead to the discovery of the structure of DNA1. First, he found that the amounts of adenine (A) and thymine (T) in DNA are almost always equal, as are the amounts of cytosine and guanine (G). Second, he observed that the proportions of A, T, G, and C in DNA vary between species. These observations suggested that DNA had a regular structure that could carry genetic information.
Rosalind Franklin: Franklin used X-ray crystallography to study the structure of DNA. Her images provided crucial information about DNA’s structure, including its helical shape and dimensions. One of her X-ray diffraction images, known as Photo 51, was critical in allowing Watson and Crick to develop their model of the DNA double helix
James Watson and Francis Crick: Using Franklin’s X-ray images along with Chargaff’s rules, Watson and Crick proposed the double helix model of DNA. They suggested that DNA is made up of two strands twisted around each other, with A always pairing with T, and C always pairing with G. This model explained how DNA could carry genetic information and how it could be copied during cell division
Discuss and interpret the work of Meselson and Stahl
- Hypotheses: Before their experiment, there were three proposed models for DNA replication:
- Semi-conservative: Each strand of the DNA molecule serves as a template for a new strand. After replication, each DNA molecule consists of one old and one new strand
- Conservative: The entire DNA molecule serves as a template for a new molecule. After replication, one DNA molecule consists of both original strands, and the other consists of two new strands
- Dispersive: DNA replication involves breakage and synthesis along the entire length of both strands, resulting in molecules that are mixtures of old and new DNA
- Experiment: Meselson and Stahl grew E. coli bacteria in a medium containing a heavy isotope of nitrogen (^15N). They then transferred these bacteria to a medium with a lighter isotope (^14N) and allowed them to replicate. They used density gradient centrifugation to separate the DNA molecules based on their densities
- Results: After one round of replication in the ^14N medium, they found that all the DNA had an intermediate density, not matching either the heavy (^15N) or light (^14N) alone. This ruled out the conservative model. After another round of replication, they found both intermediate-density DNA and light-density DNA, but no heavy-density DNA. This ruled out the dispersive model and confirmed the semi-conservative model
Explain how the AT/GC rule underlies the ability of DNA to be replicated semi conservatively
The AT/GC rule, also known as Chargaff’s rule, states that in DNA, the amount of adenine (A) is equal to the amount of thymine (T), and the amount of guanine (G) is equal to the amount of cytosine. This complementarity is crucial for the semi-conservative replication of DNA
In semi-conservative replication, each strand of the DNA molecule serves as a template for a new strand. Here’s how it works:
- The double helix unwinds, and the two strands separate
- Each strand serves as a template for a new strand
- Free nucleotides in the cell pair with the complementary bases on each template strand (A with T, and G with C) under the guidance of DNA polymerases
- The result is two DNA molecules, each composed of one original strand and one newly synthesized strand
This process ensures that the genetic information is accurately copied and passed on to the next generation of cells. The AT/GC rule is fundamental to this process because it ensures that the base pairing is always consistent, allowing for accurate replication
Describe how the synthesis of new DNA strands begins at an origin of replication
- Unwinding: The double helix unwinds at the origin of replication. Several enzymes and proteins, including topoisomerases, helicases, and gyrases (replication initiator proteins), work together to uncoil the double-stranded DNA, exposing the nitrogenous bases
- Priming: To begin synthesis, a short fragment of RNA, called a primer, must be created and paired with the template DNA strand. The primer is synthesized by an enzyme called primase
- Elongation: DNA polymerase adds a new strand of DNA by extending the 3’ end of an existing nucleotide chain, adding new nucleotides matched to the template strand, one at a time. The addition of nucleotides requires energy, which comes from the nucleotides themselves
- Leading and Lagging Strands: During DNA replication, one new strand (the leading strand) is made as a continuous piece. The other (the lagging strand) is made in small pieces called Okazaki fragments
- Completion: Finally, another enzyme called DNA ligase seals up the fragments into a continuous strand
This process ensures that each “daughter” DNA molecule is an exact copy of the “parent” DNA molecule
List the functions of helicase, topoisomerase, single-strand binding protein, primase, and DNA polymerase at the replication fork
Helicase: The enzyme unwinds the parental double helix at the replication fork
Topoisomerase: This enzyme relieves torsional strain caused by the unwinding of the double helix by helicase. It does this by causing temporary breaks in the DNA molecule
Single-Strand Binding Proteins (SSBPs): These proteins bind to the separated DNA strands to prevent them from re-forming a double helix before replication is complete. They also protect the single-stranded DNA from being degraded by nucleases
Primase: This enzyme synthesizes a short RNA primer that’s complementary to the DNA template strand. The primer provides a 3’ end for DNA polymerase to start adding nucleotides
DNA polymerase: This enzyme synthesizes a new strand of DNA based on the sequence of the template strand. It adds nucleotides to the 3’ end of the growing DNA strand
Identify the key differences in the synthesis of the leading and lagging strands
Leading:
- Synthesis occurs in the same direction as the replication fork’s movement. It is synthesized continuously in a 5’ to 3’ direction
- Requires only one primer at the origin of replication
- Synthesis of new strands is fast
- Does not require DNA ligase, as it is synthesized continuously
Lagging:
- Synthesis occurs in the opposite direction to the replication fork’s movement. It’s synthesized discontinuously in short fragments known as Okazaki fragments
- requires multiple primers, one for each Okazaki fragment
- The synthesis of new strands is slower due to the need to repeatedly create new primers
- Requires DNA ligase to join the Okazaki fragments together
Discuss the molecular structure of eukaryotic chromosomes
- DNA and Histones: At the most basic level, a chromosome is a molecule of DNA that is tightly coiled around proteins called histones. A unit of around 200 DNA base pairs wound around eight histone proteins makes up the smallest unit of DNA-packing structure, a nucleosome
- Chromatin: The DNA-histone complex is called chromatin. The beadlike, histone-DNA complex (nucleosome) and the linker DNA connecting them form a “beads on a string” structure
- 30-nm Chromatin Fiber: The nucleosomes and the linker DNA are coiled into a 30-nm chromatin fiber. This coiling further shortens the chromosome so that it’s now about 50 times shorter than the extended form
- Higher levels of compaction: In the third level of packing, a variety of fibrous proteins are used to pack the chromatin. These fibrous proteins also ensure that each chromosome in a non-dividing cell occupies a particular area of the nucleus that does not overlap with that of any other chromosomes
- Chromosome: during cell division, the chromatin condenses even further, resulting in tightly packed structures, chromosomes
Explain telomeres and the function of telomerase
- Telomeres are repetitive regions at the very ends of chromosomes that act as protective “caps.” They’re composed of repeated segments of DNA and don’t contain information needed to make proteins. They act as buffers to protect the vital coding regions. As time goes on in the process of replication, the telomeres in your body’s cells get shorter. When the telomeres are entirely gone, potentially vital regions of DNA that code for proteins will begin to be lost.
- Telomerase is an enzyme found inside our cells that adds more nucleotides to the telomeres regenerating these protective “caps” and helping the vital regions of our DNA to avoid damage. Composed of a protein component provided by the TERT gene and an RNA component; the RNA component serves as a template for adding the repetitive telomere DNA sequences to the ends of chromosomes
Replication: Circular DNA
- Initiation: An initiator protein encoded by the circular DNA nicks one strand of the double-stranded DNA molecule at a site called the double-strand origin
- Elongation: the 3’ end of the nicked strand is released to serve as a primer for DNA synthesis by DNA polymerase. Using the unnicked strand as a template, replication proceeds around the circular DNA molecule, displacing the nicked strand as single-stranded DNA
- Termination: The initiator protein makes another nick in the DNA to terminate synthesis of the first (leading) strand. RNA polymerase and DNA polymerase then replicate the single-stranded origin to make another double-stranded circle
- Completion: DNA polymerase removes the primer, replacing it with DNA, and DNA ligase joins the ends to make another molecule of double-stranded circular DNA
Replication: Linear DNA
- Initiation: replication begins at specific sequences in the DNA known as origins of replication. At these sites, various proteins and enzymes work together to unwind the double helix and make the DNA accessible for replication
- Elongation: DNA polymerase adds nucleotides to the 3’ end of a primer or a pre-existing DNA strand. Because DNA polymerase can only synthesize DNA in a 5’ to 3’ direction, one new strand (the leading strand) is synthesized continuously towards the replication fork, while the other (the lagging strand) is synthesized discontinuously away from the replication fork in short fragments known as Okazaki fragments
- Termination: When the replication forks meet, the result is two identical linear DNA molecules. Each molecule consists of one old (parental) strand and one new (daughter) strand, a process known as semi-conservative replication
- End replication problem: Because eukaryotic chromosomes are linear, they have multiple origins of replication to ensure speedy replication. DNA polymerase cannot replicate these ends, known as telomeres. To overcome this, the enzyme telomerase adds complementary bases to the 3’ end of the DNA strand at the telomeres
Replication: Antiparallel
The term “antiparallel” in the context of DNA refers to the orientation of the two strands of the DNA double helix. These strands run in opposite directions, with one strand running from 5’ to 3’ and the other from 3’ to 5’
This antiparallel structure is crucial for DNA replication. During replication, each strand serves as a template for the synthesis of a new, complementary strand. The enzyme DNA polymerase adds nucleotides to the 3’ end of the new strand, synthesizing in the 5’ to 3’ direction
Because the two strands are antiparallel, they’re replicated differently:
- Leading strand is synthesized continuously in the same direction as the replication fork
- Lagging strand is synthesized discontinuously in the opposite direction, creating short segments known as Okazaki fragments
- Fragments are later joined together by the enzyme DNA ligase to form a continuous strand. This process ensures accurate and efficient replication of DNA
Replication: 3’-5’
DNA replication primarily occurs in the 5’-3’ direction. This is because DNA polymerase can only add nucleotides to the 3’ end of the growing strand
- Leading strand is synthesized continuously in the same direction as the replication fork
- Lagging strand is synthesized discontinuously in the opposite direction, creating short segments known as Okazaki fragments
Replication: Nucleotide triphosphate
Nucleotide triphosphates play a crucial role in DNA replication. They are the molecular precursors of both DNA and RNA, which are chains of nucleotides made through the processes of DNA replication and transcription
A nucleotide triphosphate is a nucleoside containing a nitrogenous base bound to a 5-carbon sugar (either ribose or deoxyribose), with three phosphate groups bound to the sugar. For example, deoxyadenosine triphosphate (dATP) is a nucleotide used in cells for DNA synthesis, as a substrate of DNA polymerase
The three phosphates are joined to each other by high-energy bonds. The cleavage of these bonds during the polymerization reaction releases the free energy needed to drive the incorporation of each nucleotide into the growing DNA chain. This is why DNA replication is performed in the 5’-3’ direction
Replication: Semiconservative
Confirmed by the Meselson-Stahl experiments in 1958:
- They labeled the DNA of bacteria across generations using isotopes of nitrogen
-In semiconservative replication, two strands of DNA unwind from each other, and each acts as a template for the synthesis of a new complementary strand. This results in two DNA molecules, each composed of one original strand and one new strand
Replication: Replication fork
A region where the DNA double helix has been unwound and separated, creating an area where DNA polymerases and other enzymes can use each strand as a template to synthesize
Replication: Helicase
Unwinds the double helix of DNA so that it can be replicated
- breaks hydrogen bonds, separating the leading and lagging strands, allowing replication to occur
Replication: DNA polymerase
- Add nucleotides one by one to the growing DNA chain, incorporating only those that are complementary to the template
- They can only add nucleotides to the 3’ end of a DNA strand, synthesizing DNA in the 5’ to 3’ direction
- They can’t start making a DNA chain from scratch, but require a pre-existing chain or short stretch of nucleotides called a primer
- They proofread, or check their work, removing the vast majority of “wrong” nucleotides that are accidentally added to the chain
- The addition of nucleotides require energy. This energy comes from the nucleotides themselves, which have three phosphates attached to them
Replication: DNA ligase
- catalyzes the formation of a phosphodiester bond. The enzyme joins the 3’ hydroxyl group of one nucleotide with the 5’ phosphate end of another nucleotide
- Plays a role in repairing single-strand breaks in duplex DNA in living organisms
- Ligase’s job is to join fragments of newly synthesized DNA to form a seamless strand (particularly important on the lagging strand, where DNA is synthesized discontinuously as Okazaki fragments
- Two ATP molecules are consumed for each phosphodiester bond formed
Replication: DNA primase
- Catalyzes the synthesis of a short RNA segment called a primer. This primer is complementary to a single-stranded DNA template
- Role in Replication: Since DNA polymerases can only recognize and elongate double-stranded sequences, the role of DNA primase in DNA replication is to synthesize a short RNA segment (i.e., a primer) complementary to the ssDNA template. This provides a double-stranded fragment for the DNA polymerase to recognize, thus initiating replication
- The primer is a short single strand of RNA ranging from 8 to 12 nucleotides, complementary to the starting bases of the leading strand of DNA
Replication: Primer
- Primers are short strands of RNA and has to be synthesized by an enzyme called primase
- Added before DNA polymerase can begin a complementary strand
- After the DNA polymerase adds nucleotides following the binding to the RNA primer and synthesizes the whole strand, the RNA strands must be removed accurately and replaced with DNA nucleotides forming a gap region known as a nick that is filled in using an enzyme called ligase
- primer is a short, single strand of RNA ranging from 8 to 12 nucelotides, complementary to the starting bases of the leading strand of DNA
Replication: Topoisomerase
- Allows DNA to change its topology (shape). In bacteria, DNA exists in a single circular chromosomes. However, in multicellular life, it’s bundled in X-shaped chromosomes
- During DNA replication, both the chromosomal supercoil and the double helix must be unwound to make room for the addition of a new strand to the double-stranded molecule. This adds pressure to the DNA that’s coiled and also has the potential to create a messy tangle of uncoiled DNA
- Helps prevents tangles by creating tiny cuts in the DNA strand to help it unravel the extra tension and make room for the replication machinery
- Assists in creating the replication fork by cutting the double-helix to allow it to rotate and then reconnecting the ends of the relaxed DNA
- prevents the DNA double helix ahead of the replication fork from getting too tightly wound as DNA is opened up. It acts by making temporary nicks in the helix to release tension, then sealing the nicks to avoid permanent damage
