7.1 Flashcards
series of experiments to prove that DNA was the genetic material
Alfred Hershey and Martha Chase in 1952
Viruses (T2 bacteriophage) were grown in one of two isotopic mediums in order to radioactively label a specific viral component
- Viruses grown in radioactive sulfur (35S) had radiolabelled proteins (sulfur is present in proteins but not DNA)
- Viruses grown in radioactive phosphorus (32P) had radiolabeled DNA (phosphorus is present in DNA but not proteins)
virus and bacteria were…
The viruses were then allowed to infect a bacterium (E. coli) and then the virus and bacteria were separated via centrifugation
- The larger bacteria formed a solid pellet while the smaller viruses remained in the supernatant
Hershey and Chase demonstrated that
he bacterial pellet was found to be radioactive when infected by the 32P–viruses (DNA) but not the 35S–viruses (protein)
- This demonstrated that DNA, not protein, was the genetic material because DNA was transferred to the bacteria
Rosalind Franklin and Maurice Wilkins used a method of X-ray diffraction to investigate the structure of DNA
- DNA was purified and then fibres were stretched in a thin glass tube (to make most of the strands parallel)
- The DNA was targeted by a X-ray beam, which was diffracted when it contacted an atom
- The scattering pattern of the X-ray was recorded on a film and used to elucidate details of molecular structure
From the scattering pattern produced by a DNA molecule, certain inferences could be made about its structure
- Composition: DNA is a double stranded molecule
- Orientation: Nitrogenous bases are closely packed together on the inside and phosphates form an outer backbone
- Shape: The DNA molecule twists at regular intervals (every 34 Angstrom) to form a helix (two strands = double helix)
Franklin’s x-ray diffraction experiments demonstrated that…
DNA helix is both tightly packed and regular in structure
- Phosphates (and sugars) form an outer backbone and nitrogenous bases are packaged within the interior
Chargaff had also demonstrated that DNA is composed of…
an equal number of purines (A + G) and pyrimidines (C + T)
- This indicates that these nitrogenous bases are paired (purine + pyrimidine) within the double helix
- In order for this pairing between purines and pyrimidines to occur, the two strands must run in antiparallel directions
When Watson & Crick were developing their DNA model, they discovered that…
an A–T bond was the same length as a G–C bond
- Adenine and thymine paired via two hydrogen bonds, whereas guanine and cytosine paired via three hydrogen bonds
- If the bases were always paired this way, then this would describe the regular structure of the DNA helix (shown by Franklin)
Consequently, DNA structure suggests two mechanisms for DNA replication:
- Replication occurs via complementary base pairing (adenine pairs with thymine, guanine pairs with cytosine)
- Replication is bi-directional (proceeds in opposite directions on the two strands) due to the antiparallel nature of the strands
Helicase
- Helicase unwinds and separates the double-stranded DNA by breaking the hydrogen bonds between base pairs
- This occurs at specific regions (origins of replication), creating a replication fork of two strands running in antiparallel directions
DNA Gyrase
- DNA gyrase reduces the torsional strain created by the unwinding of DNA by helicase
- It does this by relaxing positive supercoils (via negative supercoiling) that would otherwise form during the unwinding of DNA
Single Stranded Binding (SSB) Proteins
- SSB proteins bind to the DNA strands after they have been separated and prevent the strands from re-annealing
- These proteins also help to prevent the single stranded DNA from being digested by nucleases
- SSB proteins will be dislodged from the strand when a new complementary strand is synthesised by DNA polymerase III
DNA Primase
- DNA primase generates a short RNA primer (~10–15 nucleotides) on each of the template strands
- The RNA primer provides an initiation point for DNA polymerase III, which can extend a nucleotide chain but not start one
DNA Polymerase III
- Free nucleotides align opposite their complementary base partners (A = T ; G = C)
- DNA pol III attaches to the 3’-end of the primer and covalently joins the free nucleotides together in a 5’ → 3’ direction
- As DNA strands are antiparallel, DNA pol III moves in opposite directions on the two strands
-On the leading strand, DNA pol III is moving towards
the replication fork and can synthesise continuously
- On the lagging strand, DNA pol III is moving away
from the replication fork and synthesises in pieces
(Okazaki fragments)
DNA Polymerase I
- As the lagging strand is synthesised in a series of short fragments, it has multiple RNA primers along its length
- DNA pol I removes the RNA primers from the lagging strand and replaces them with DNA nucleotides
DNA Ligase
- DNA ligase joins the Okazaki fragments together to form a continuous strand
- It does this by covalently joining the sugar-phosphate backbones together with a phosphodiester bond
DNA polymerase cannot…
Initiate replication, it can only add new nucleotides to an existing strand
for DNA replication to occur…
an RNA primer must first be synthesised to provide an attachment point for DNA polymerase
DNA polyermase occurs…
nucleotides to the 3’ end of a primer, extending the new chain in a 5’ → 3’ direction
- Free nucleotides exist as deoxynucleoside triphosphates (dNTPs) – they have 3 phosphate groups
- DNA polymerase cleaves the two additional phosphates and uses the energy released to form a phosphodiester bond with the 3’ end of a nucleotide chain
Leading versus Lagging Strands
Because double-stranded DNA is antiparallel, DNA polymerase must move in opposite directions on the two strands
On the leading strand…
DNA polymerase is moving towards the replication fork and so can copy continuously
On the lagging strand…
DNA polymerase is moving away from the replication fork, meaning copying is discontinuous
As DNA polymerase is moving away from helicase…
it must constantly return to copy newly separated stretches of DNA
the lagging strand is…
copied as a series of short fragments (Okazaki fragments), each preceded by a primer
the primers are replaced with…
DNA bases and the fragments joined together by a combination of DNA pol I and DNA ligase
DNA sequencing refers to…
the process by which the base order of a nucleotide sequence is elucidated
The most widely used method for DNA sequencing involved the use of…
chain-terminating dideoxynucleotides
dideoxynucleotides
- Dideoxynucleotides (ddNTPs) lack the 3’-hydroxyl group necessary for forming a phosphodiester bond
- Consequently, ddNTPs prevent further elongation of a nucleotide chain and effectively terminate replication
- The resulting length of a DNA sequence will reflect the specific nucleotide position at which the ddNTP was incorporated
- For example, if a ddGTP terminates a sequence after
8 nucleotides, then the 8th nucleotide in the
sequence is a cytosine
- For example, if a ddGTP terminates a sequence after
Sequencing
Dideoxynucleotides can be used to determine DNA sequence using the Sanger method
Sanger Sequencing method
- Four PCR mixes are set up, each containing stocks of normal nucleotides plus one dideoxynucleotide (ddA, ddT, ddC or ddG)
- As a typical PCR will generate over 1 billion DNA molecules, each PCR mix should generate all the possible terminating fragments for that particular base
- When the fragments are separated using gel electrophoresis, the base sequence can be determined by ordering fragments according to length
- If a distinct radioactive or fluorescently labelled primer is included in each mix, the fragments can be detected by automated sequencing machines
- If the Sanger method is conducted on the coding strand (non-template strand), the resulting sequence elucidated will be identical to the template strand
The vast majority of the human genome is comprised of…
non-coding DNA (genes only account for ~ 1.5% of the total sequence)
non-coding DNA was historically referred to as
‘junk DNA’, these non-coding regions are now recognised to serve other important functions
examples of ‘junk DNA’
satellite DNA, telomeres, introns, ncRNA genes and gene regulatory sequences
DNA profiling is a technique by which…
individuals can be identified and compared via their respective DNA profiles
short tandem repeats (STRs)
Within the non-coding regions of an individual’s genome there exists satellite DNA – long stretches of DNA made up of repeating elements
Tandem repeats can be excised using
restriction enzymes and then separated with gel electrophoresis for comparison
As individuals will likely have different numbers of repeats at a given satellite DNA locus, they will generate
unique DNA profiles
Longer repeats will generate…
longer fragments (and vice versa)
in eukaryotic organisms, the DNA is packaged with…
histone proteins to create a compacted structure called a nucleosome
Nucleosomes help to…
supercoil the DNA, resulting in a greatly compacted structure that allows for more efficient storage
Supercoiling helps to…
protect the DNA from damage and also allows chromosomes to be mobile during mitosis and meiosis
nucleosome
The DNA is complexed with eight histone proteins (an octamer) to form a complex
chromatosomes
Nucleosomes are linked by an additional histone protein (H1 histone) to form a string of
30 nm fibre
These then coil to form a solenoid structure (~6 chromatosomes per turn) which is condensed to form a
chromatin
These fibres then form loops, which are compressed and folded around a protein scaffold to form
chromosomes
Chromatin will then supercoil during cell division to form chromosomes that are visible (when stained) under microscope
A nucleosome consists of…
a molecule of DNA wrapped around a core of eight histone proteins (an octamer)
nucleosome
- The negatively charged DNA associates with positively charged amino acids on the surface of the histone proteins
- The histone proteins have N-terminal tails which extrude outwards from the nucleosome
- During chromosomal condensation, tails from adjacent histone octamers link up and draw the nucleosomes closer together
