Topic 7.1 DNA Structure and Replication Flashcards
Proof of DNA being the genetic material
In 1952, Alfred Hershey and Martha Chase conducted a series of experiments to prove that DNA was the genetic material
- 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)
Structure of DNA
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
Inferences made about the DNA 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)
DNA Replication (HL)
Helicase
- Helicase unwinds and separates the double-stranded DNA by breaking the hydrogen bonds between base pairs
DNA Gyrase
- DNA gyrase reduces the torsional strain created by the unwinding of DNA by helicase
Single Stranded Binding (SSB) Proteins
- SSB proteins bind to the DNA strands after they have been separated and prevent the strands from re-annealing
DNA Primase
- DNA primase generates a short RNA primer (~10–15 nucleotides) on each of the template
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
DNA Polymerase I
- 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
Leading vs Lagging Strand
- 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
- As DNA polymerase is moving away from helicase, it must constantly return to copy newly separated stretches of DNA
Sequencing using the Sanger 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
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
Non-coding DNA
- Historically referred to as ‘junk DNA’, these non-coding regions are now recognized to serve other important functions
- Examples include satellite DNA, telomeres, introns, ncRNA genes and gene regulatory sequences
Description of examples of non-coding DNA
S - Satellite DNA - Tandem repeating sequences of DNA, commonly used for DNA profiling
T - Telomeres - Regions of repetitive DNA at the end of a chromosome, also prevents chromosomal deterioration
I - Introns - Non-coding sequences within genes, are removed by RNA splicing prior to the formation of mRNA
N - Non-coding RNA genes - Codes for RNA molecules that are not translated into proteins, examples include tRNA
G - Gene regulatory sequences - Sequences that are involved in the process of transcription, includes promoters, enhancers and silencers
DNA Profiling
a technique by which individuals can be identified and compared via their respective DNA profiles
How does DNA profiling work?
- Within the non-coding regions of an individual’s genome there exists satellite DNA – long stretches of DNA made up of repeating elements called short tandem repeats (STRs)
- 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
Organization of Eukaryotic DNA
- The DNA is complexed with eight histone proteins (an octamer) to form a complex called a nucleosome
- Nucleosomes are linked by an additional histone protein (H1 histone) to form a string of chromatosomes
- These then coil to form a solenoid structure (~6 chromatosomes per turn) which is condensed to form a 30 nm fibre
- These fibres then form loops, which are compressed and folded around a protein scaffold to form chromatin
- Chromatin will then supercoil during cell division to form chromosomes that are visible (when stained) under microscope
