1
Q
What was the Griffiths experiment?
A
- Studied two strains of the bacterium Streptococcus pneumoniae: S (smooth) strain can cause pneumonia in mice; it is pathogenic because the cells have an outer capsule that protects them from an animal’s immune system. Cells of the R (rough) strain lack a capsule and are nonpathogenic
- Living S cells > mouse dies
- Living R cells > mouse healthy
- Heat-killed S cells > mouse healthy
- Mixture of heat-killed S cells and living R cells > mouse dies
- when he killed the pathogenic bacteria with heat and then mixed the cell remains with living bacteria of the nonpathogenic strain, some of the living cells became pathogenic; this newly acquired trait of pathogenicity was inherited by all the descendants of the transformed bacteria
- some chemical component of the dead pathogenic cells caused this heritable change, although the identity of the substance was not known; Griffith called the phenomenon transformation, now defined as a change in genotype and phenotype due to the assimilation of external DNA by a cell
2
Q
What was the primary belief of hereditary material before the discovery of DNA?
A
- Primary belief was that proteins were the hereditary molecule
- Variety (20 different amino acids vs 4 bases)
3
Q
What are viruses and bacteriophages?
A
- a virus is an infectious particle incapable of replicating outside of a cell, consisting of an RNA or DNA genome surrounded by a protein coat (capsid) and for some viruses, a membranous envelop (to produce more viruses, a virus must infect a cell and take over the cell’s metabolic machinery)
- bacteriophages are viruses that infect bacteria
4
Q
What was the Hershey and Chase experiment?
A
- performed experiments showing that DNA is the genetic material of a phage known as T2 which infect the bacterium Escherichia coli
- At that time, biologists already knew that T2, like many other phages, was composed almost entirely of DNA and protein knew that the T2 phage could quickly turn an E. coli cell into a T2-producing factory, but which viral component, protein or DNA, was responsible?
- Batch 1: they used a radioactive isotope of sulphur to tag protein in one batch of T2 because protein, but not DNA, contains sulphur
- Batch 2: a radioactive isotope of phosphorus was used to tag DNA in a second batch. In a similar way, the atoms of radioactive phosphorus labelled only the DNA, not the protein, because nearly all the phage’s phosphorus is in its DNA.
1) nonradioactive E.coli cells were infected with the protein-labelled and DNA-labelled batches of T2
2) this mixture of E. coli and radioactively-labelled phages were agitated in a blender to free phage parts outside the bacteria from the cells
3) centrifuged (rapidly rotating container that applies centrifugal force and separates liquids of different densities) the mixture so that bacteria formed a pellet (bacterial cells and contents) at the bottom of the test tube and free phage and phage parts remained suspended in the liquid
- Batch 2: a radioactive isotope of phosphorus was used to tag DNA in a second batch. In a similar way, the atoms of radioactive phosphorus labelled only the DNA, not the protein, because nearly all the phage’s phosphorus is in its DNA.
- batch 1: radioactivity (phage protein) found in liquid ( meaning radioactivity remained outside the cells)
- batch 2: radioactivity (phage DNA) found in pellet (meaning radioactivity was found inside the cells); cells containing radioactive phage DNA released new phages with some radioactive phosphorus
- concluded that the DNA injected by the phage must be the molecule carrying the genetic information
5
Q
What is Chargaff’s rule?
A
1) the percentages of A and T bases are roughly equal, as are those of G and C bases (The amount of purines = to amount of pyrimidines)
2) DNA base composition varies between species (the percentage of each pair (A/T or C/G) varied from species to species)
6
Q
How was the structure of DNA discovered?
A
- Watson saw an X-ray diffraction image of DNA produced by Rosalind Franklin, suggesting the width of the helix and the spacing of the nitrogenous bases along it
- The pattern in this photo implied that the helix was made up of two strands, contrary to a three-stranded model that Linus Pauling had proposed
- Watson and Crick began building models of a double helix that would conform to the X-ray measurements and what was then known about the chemistry of DNA, including Chargaff’s rule of base equivalences
- Having also read an unpublished annual report summarizing Franklin’s work, they knew she had concluded that the sugar-phosphate backbones were on the outside of the DNA molecule, contrary to their working model
- At first, Watson imagined that the bases paired like with like—for example, A with A and C with C. But this model did not fit the X-ray data, which suggested that the double helix had a uniform diameter of 2nm; Pairing a purine with a pyrimidine is the only combination that results in a uniform diameter for the double helix
7
Q
What was Watson & Crick’s semi-conservative model?
A
– type of DNA replication in which the replicated double helix consists of one old strand, derived from the parental molecule, and one newly made strand; each parental molecule functions as a template for synthesis of a new, complimentary strand
8
Q
What is the conservative and dispersive model?
A
- conservative: the two parental strands reassociate after acting as templates for new strands, thus restoring the parental double helix
- dispersive: each strand of both daughter molecules contains a mixture of old and newly synthesized DNA
9
Q
What was the Meselson and Stahl study that distinguished between the three models?
A
- E. coli bacteria cultured in a medium with 15N (heavy isotope)
- they then transferred this bacteria to a medium with 14N (lighter isotope)
- a sample was taken after the first DNA replication; another sample was taken after the second replication
- They extracted DNA from the bacteria in the samples and then centrifuged each DNA sample to separate DNA of different densities; the DNA settles at a particular level within the liquid and that indicates its weight (the DNA in the 14N will be lighter and thus float higher than the DNA in the 15N)
- if it was the conservative model, you would expect to see two bands: a set of 15N DNA floating at the bottom (parent DNA) and a set of 14N DNA floating at the top (new DNA)
- The first replication in the 14N medium produced one band of hybrid (15N−14N) DNA of mid-weight floating in the middle; this result eliminated the conservative model (however, this would also happen in the dispersive model so they checked again after second replication)
- In a semi-conservative model, after a second replication, you would expect to get a two bands of hybrid DNA and another two bands of light DNA floating at the top
- in a dispersive model, after a second replication, you would expect to get only one band of light (14N) DNA
- Instead, what they found was that the second replication produced both light and hybrid DNA, a result that refuted the dispersive model and supported the semiconservative model
10
Q
What is DNA made up of?
A
- a polymer of four nucleotides, each having three components: a nitrogenous (nitrogen-containing) base, a pentose sugar called deoxyribose, and a phosphate group
- nitrogenous base: purines (two organic rings: adenine & guanine) and pyrimidines (one organic ring: thymine & cytosine)
11
Q
What is the directionality of a polynucleotide strand?
A
– A polynucleotide strand has directionality, from the 5′ end (with the free phosphate group) to the 3′ end (with the free ―OH group of the sugar). 5′ and 3′ refer to the numbers assigned to the carbons in the sugar ring
12
Q
What chemical bonds make a polynucleotide and DNA strand?
A
- phosphodiester bonds between nucleotides (phosphate group on 5’ end and OH group on 3’ end)
- Hydrogen bonds between nitrogenous base pairs (hold two strands of DNA together)
13
Q
How many hydrogen bonds can the base pairs hold?
A
– Adenine can form two hydrogen bonds with thymine and only thymine (weaker); guanine forms three hydrogen bonds with cytosine and only cytosine (stronger)
14
Q
What is the origin of replication and replication fork?
A
- origin of replication: short stretches of DNA that have a specific sequence of nucleotides and where replication of a DNA molecule begins; proteins that initiate DNA replication recognize this sequence and attach to the DNA, separating the two strands and opening up a replication “bubble”; replication of DNA then proceeds in both directions until the entire molecule is copied (this segment mostly contains AT pairs because they have two hydrogen bonds so it is weaker)
- replication fork: a Y-shaped region at each end of a replication bubble where the parental strands of DNA are being unwound
15
Q
How does origin of replication differ in eukaryotes and prokaryotes ?
A
- In contrast to a bacterial chromosome, a eukaryotic chromosome may have hundreds or even a few thousand replication origins
- As in bacteria, eukaryotic DNA replication proceeds in both directions from each origin.
16
Q
What are helicases?
A
– enzymes that untwist the double helix of DNA at replication forks, separating the two strands and making them available as template strands
17
Q
What are single-strand binding proteins?
A
– a protein that binds to the unpaired DNA strands during DNA replication, stabilizing them and holding them apart while they serve as templates for the synthesis of complementary strands of DNA
18
Q
What are topoisomerase?
A
- the untwisting of the double helix causes tighter twisting and strain ahead of the replication fork
- Topoisomerase is an enzyme that helps relieve this strain by breaking, swivelling, and rejoining DNA strands
19
Q
What is a primer and primase?
A
- the enzymes that synthesize DNA cannot initiate the synthesis of a polynucleotide; they can only add DNA nucleotides to the end of an already existing chain that is base-paired with the template strand; this initial nucleotide chain is actually a short stretch of RNA called a primer, synthesized by the enzyme primase
- Primase starts a complementary RNA chain with a single RNA nucleotide, and adds RNA nucleotides one at a time, using the parental DNA strand as a template. The completed primer, generally 5–10 nucleotides long, is thus base-paired to the template strand. The new DNA strand will start from the 3′ end of the RNA primer
20
Q
What are DNA polymerases III?
A
- Enzymes that catalyze the synthesis of new DNA by adding nucleotides to the 3′ end of a preexisting chain which is the primer (can only add nucleotides to the 3’ end and never 5’ end, therefore DNA grows from the 5’ to 3’ end)
- Most DNA polymerases require a primer and a DNA template strand, along which complementary DNA nucleotides are lined up
21
Q
What is dATP and how does it differ from ATP?
A
- dATP is the adenine nucleotide added to a growing DNA strand; consists of a sugar attached to a base and to three phosphate groups
- The only difference between the ATP of energy metabolism and dATP, is the sugar component, which is deoxyribose in the building block of DNA but ribose in ATP (ATP has a ribose sugar, three phosphate groups and adenine base)
- Like ATP, the nucleotides used for DNA synthesis are chemically reactive, partly because their triphosphate tails have an unstable cluster of negative charged that constantly repel each other
22
Q
Where does the energy to drive the DNA polymerization reaction come from?
A
- As DNA polymerase catalyzes each dehydration reaction (phosphodiester bond) that joins a monomer to the growing end of a DNA strand, two phosphate groups are lost as a molecule of pyrophosphate
- Subsequent hydrolysis of the pyrophosphate to two molecules of inorganic phosphate is a coupled exergonic reaction that helps drive the polymerization reaction
- Subsequent hydrolysis of the pyrophosphate to two molecules of inorganic phosphate is a coupled exergonic reaction that helps drive the polymerization reaction
23
Q
What is the leading strand?
A
- the new complimentary DNA strand synthesized by DNA pol III continuously along the template strand toward the replication fork in the mandatory 5′ → 3′ direction
- Only one primer is required for DNA pol III to synthesize the entire leading strand
24
Q
What is the lagging strand?
A
- a discontinuously synthesized DNA strand (by DNA pol III) that elongates (synthesized) in the 5′ → 3′ direction away from the replication fork
- In contrast to the leading strand, which elongates continuously, the lagging strand is synthesized discontinuously, as a series of segments called Okazaki fragments
- this is because the fork has to expose enough of the strand for the primase to lay down a primer; this continues along the length of the lagging strand, waiting for the fork to expose enough of the strand to make primers
- Whereas only one primer is required on the leading strand, each Okazaki fragment on the lagging strand must be primed separately
25
What is DNA polymerase I and DNA ligase?
- - After DNA pol III forms an Okazaki fragment, it detaches when it reaches the other fragment's primer; DNA polymerase I replaces the RNA nucleotides of the adjacent primer with DNA nucleotides
- - But DNA pol I cannot join the final nucleotide of this replacement DNA segment to the first DNA nucleotide of the adjacent Okazaki fragment. Another enzyme, DNA ligase, accomplishes this task, joining the sugar-phosphate backbones of all the Okazaki fragments into a continuous DNA strand
26
Summary of all proteins in DNA replication
- - Helicase: unwinds parental double helix at replication forks
- - Single strand binding protein: binds to and stabilizes single-stranded DNA until it is used as a template
- - Topoisomerase: relieves overwinding strain ahead of replication forks by breaking, swivelling, and rejoining DNA strands
- - Primase: synthesizes an RNA primer at 5′ end of leading strand and at 5′ end of each Okazaki fragment of lagging strand
- - DNA pol III: Using parental DNA as a template, synthesizes new DNA strand by adding nucleotides to an RNA primer or a preexisting DNA strand
- - DNA pol I: removes RNA nucleotides of primer from 5′ end and replaces them with DNA nucleotides
- - DNA ligase: joins Okazaki fragments of lagging strand; on leading strand, joins 3′ end of DNA that replaces primer to rest of leading strand DNA
27
What is the DNA replication machine?
- - the various proteins that participate in DNA replication actually form a single large complex, a “DNA replication machine"
- - Many protein-protein interactions facilitate the efficiency of this complex (by interacting with other proteins at the fork, primase apparently acts as a molecular brake, slowing progress of the replication fork and coordinating the placement of primers and the rates of replication on the leading and lagging strands)
- - the DNA replication complex may not move along the DNA; rather, the DNA may move through the complex during the replication process
- - In eukaryotic cells, multiple copies of the complex, perhaps grouped into “factories,” may be anchored to the nuclear matrix; experimental evidence in some types of cells supports a model in which two DNA polymerase molecules, one on each template strand, “reel in” the parental DNA and extrude newly made daughter DNA molecules
28
How is DNA proofread?
- - during DNA replication, many DNA polymerases proofread each nucleotide against its template as soon as it is covalently bonded to the growing strand. Upon finding an incorrectly paired nucleotide, the polymerase removes the nucleotide and then resumes synthesis
- - Incorrectly paired or altered nucleotides can also arise after replication; DNA molecules are constantly subjected to potentially harmful chemical and physical agent
- - In addition, DNA bases may undergo spontaneous chemical changes under normal cellular conditions. However, these changes in DNA are usually corrected before they become permanent changes—mutations—perpetuated through successive replications
- - Each cell continuously monitors and repairs its genetic material which is why many different DNA repair enzymes have evolved
29
What is a mismatch repair?
- - Mismatched nucleotides sometimes evade proofreading by a DNA polymerase
- - In mismatch repair, other enzymes remove and replace incorrectly paired nucleotides that have resulted from replication errors; researchers highlighted the importance of such repair enzymes when they found that a hereditary defect in one of them is associated with a form of colon cancer.
30
What is the nucleotide excision repair?
- - Most cellular systems for repairing incorrectly paired nucleotides, whether they are due to DNA damage or to replication errors, use a mechanism that takes advantage of the base-paired structure of DNA
- - a nuclease is an enzyme that cuts the damaged segment of DNA or RNA, either by removing one or few bases or hydrolyzing the DNA or RNA completely into its component nucleotides
- - the resulting gap is then filled in with nucleotides, using the undamaged strand as a template; the enzymes involved in filling the gap are a DNA polymerase and DNA ligase
- - this DNA repair system is called nucleotide excision repair
31
What are thymine dimers and what do they cause?
- - the covalent linking of thymine bases that are adjacent on a DNA strand caused by UV rays from sunlight; such thymine dimers cause the DNA to buckle and interfere with DNA replication
- - the importance of repairing this kind of damage is underscored by a disorder called xeroderma pigmentosum (XP), which in most cases is caused by an inherited defect in a nucleotide excision repair enzyme. Individuals with XP are hypersensitive to sunlight; mutations in their skin cells caused by ultraviolet light are left uncorrected, often resulting in skin cancer
32
What are mutations?
- - Once a mismatched nucleotide pair is replicated, the sequence change is permanent in the daughter molecule that has the incorrect nucleotide as well as in any subsequent copies; this permanent change in the DNA sequence is called a mutation
- - mutations are the original source of the variation on which natural selection operates during evolution and are ultimately responsible for the appearance of new species
33
Why do repeated rounds of replication produce shorter and shorter DNA molecules with uneven staggered ends?
- - For linear DNA, such as the DNA of eukaryotic chromosomes, the usual replication machinery cannot complete the 5′ ends of daughter DNA
- - Even if an Okazaki fragment can be started with an RNA primer hydrogen-bonded to the very end of the template strand, once that primer is removed, it cannot be replaced with DNA because there is no 3′ end available for nucleotide addition; As a result, repeated rounds of replication produce shorter and shorter DNA molecules with uneven (“staggered”) ends.
- - Most prokaryotes have a circular chromosome, with no ends, so the shortening of DNA does not occur
34
What are telomeres?
- - the tandemly repetitive DNA at the end of a eukaryotic chromosome's DNA molecule; telomeres protect the organism's genes from being eroded during successive rounds of replication
- - Telomeres do not contain genes; instead, the DNA typically consists of multiple repetitions of one short nucleotide sequence. In each human telomere, for example, the six-nucleotide sequence TTAGGG is repeated between 100 and 1000 times.
- - two protective functions: First, specific proteins associated with telomeric DNA prevent the staggered ends of the daughter molecule from activating the cell’s systems for monitoring DNA damage, leading to cell arrest/death. Second, telomeric DNA acts as a kind of buffer zone that provides some protection against the organism’s genes shortening (postpone erosion of genes), somewhat like how the plastic-wrapped ends of a shoelace slow down its unravelling
- - Thus, as expected, telomeric DNA tends to be shorter in dividing somatic cells of older individuals and in cultured cells that have divided many times; proposed that shortening of telomeres is somehow connected to the aging process of certain tissues and even to aging of the organism as a whole
35
What is telomerase?
- - An enzyme called telomerase catalyzes the lengthening of telomeres in eukaryotic germ cells, thus restoring their original length and compensating for the shortening that occurs during DNA replication
- - This enzyme contains its own RNA molecule that it uses as a template to artificially “extend” the leading strand, allowing the lagging strand to maintain a given length; The activity of telomerase in germ cells results in telomeres of maximum length in the zygote
- - Telomerase is not active in most human somatic cells, but its activity varies from tissue to tissue
36
How do shortening of telomeres protect organisms from cancer?
- - Normal shortening of telomeres may protect organisms from cancer by limiting the number of divisions that somatic cells can undergo
- - Cells from large tumours often have unusually short telomeres, as we would expect for cells that have undergone many cell divisions. Further shortening would presumably lead to self-destruction of the tumour cells
- - Telomerase activity is abnormally high in cancerous somatic cells, suggesting that its ability to stabilize telomere length may allow these cancer cells to persist; researchers have studied inhibition of telomerase as a possible cancer therapy
BIOL1103
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