7.1 DNA Structure Flashcards
What problem did scientists in the mid-twentieth century relating to genetic material?
In the mid-twentieth century, scientists were still unsure as to whether DNA or protein was the genetic material of the cell
Why did scientists had trouble deciding whether protein or DNA were the genetic material?
It was known that some viruses consisted solely of DNA and a protein coat and could transfer their genetic material into hosts
Who carried out an experiment to determine whether DNA or protein is the genetic material of the cell?
In 1952, Alfred Hershey and Martha Chase conducted a series of experiments to prove that DNA was the genetic material
- What was grown to prove 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
- What were the 2 conditions in which the viruses were grown?
Viruses grown in radioactive sulfur (35S) had radiolabelled proteins
Viruses grown in radioactive phosphorus (32P) had radiolabeled DNA
Why were some of the viruses grown in sulfur?
h&c
sulfur is present in proteins but not DNA
Why were some of the viruses grown in phosphorus?
h&c
phosphorus is present in DNA but not proteins
- What were the viruses then allowed to do? h & c
The viruses were then allowed to infect a bacterium (E. coli) and then the virus and bacteria were separated via centrifugation
- What happened during the centrifugation? h&c
The larger bacteria formed a solid pellet while the smaller viruses remained in the supernatant
- What were the results? h&c
The bacterial pellet was found to be radioactive when infected by the 32P–viruses (DNA) but not the 35S–viruses (protein)
- What could they conclude? h&c
This demonstrated that DNA, not protein, was the genetic material because DNA was transferred to the bacteria
Who researched the structure of DNA? What did they use?
Rosalind Franklin and Maurice Wilkins used a method of X-ray diffraction to investigate the structure of DNA
- What is the first step of x-ray diffraction?
DNA was purified and then fibres were stretched in a thin glass tube (to make most of the strands parallel)
- What is concentrated on the DNA? x-ray
The DNA was targeted by a X-ray beam, which was diffracted when it contacted an atom
- What does the x-ray diffraction show?
The scattering pattern of the X-ray was recorded on a film and used to elucidate details of molecular structure
What 3 things can be determined from the x-ray diffraction?
composition
orientation
shape
What was concluded about the composition of DNA from the x-ray diffraction?
DNA is a double stranded molecule
What was concluded about the orientation of DNA from the x-ray diffraction?
Nitrogenous bases are closely packed together on the inside and phosphates form an outer backbone
What was concluded about the shape of DNA from the x-ray diffraction?
The DNA molecule twists at regular intervals (every 34 Angstrom) to form a helix (two strands = double helix)
What did Franklin’s research conclude?
Franklin’s x-ray diffraction experiments demonstrated that the DNA helix is both tightly packed and regular in structure
What is the structure of DNA?
Phosphates (and sugars) form an outer backbone and nitrogenous bases are packaged within the interior
What did Chargriff determine?
Chargaff had also demonstrated that DNA is composed of an equal number of purines (A + G) and pyrimidines (C + T)
What can be inferred from Chargraff’s findings?
This indicates that these nitrogenous bases are paired (purine + pyrimidine) within the double helix
How did Chargraff’s findings help in determining the directions of the DNA strands?
In order for this pairing between purines and pyrimidines to occur, the two strands must run in antiparallel directions
What information about the hydrogen bonds was concluded by Watson and Crick?
When Watson & Crick were developing their DNA model, they discovered that an A–T bond was the same length as a G–C bond
How many bonds are formed between A and T, G and C?
Adenine and thymine paired via two hydrogen bonds, whereas guanine and cytosine paired via three hydrogen bonds
What does the DNA structure suggest about the 2 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
What type of process is DNA replication?
DNA replication is a semi-conservative process that is carried out by a complex system of enzymes
What is helicase’s role in DNA replication?
Helicase unwinds and separates the double-stranded DNA by breaking the hydrogen bonds between base pairs
Where does helicase unwind DNA?
This occurs at specific regions (origins of replication), creating a replication fork of two strands running in antiparallel directions
What is the role of DNA gyrase?
DNA gyrase reduces the torsional strain created by the unwinding of DNA by helicase
How does DNA gyrase reduce the torsional strain?
It does this by relaxing positive supercoils (via negative supercoiling) that would otherwise form during the unwinding of DNA
What 7 proteins are involved in DNA replication?
helicase DNA gyrase SSB Proteins (single-stranded binding proteins) DNA primase DNA polymerase III DNA polymerase I DNA Ligase
What is the role of SSB’s in DNA replication?
SSB proteins bind to the DNA strands after they have been separated and prevent the strands from re-annealing
Apart from separating the DNA strands, what is the role of SSB’s?
These proteins also help to prevent the single stranded DNA from being digested by nucleases
What happens to SSBs once DNA replication is finished?
SSB proteins will be dislodged from the strand when a new complementary strand is synthesised by DNA polymerase III
What is the role of DNA primase?
DNA primase generates a short RNA primer (~10–15 nucleotides) on each of the template strands
What is the role of RNA primer?
The RNA primer provides an initiation point for DNA polymerase III, which can extend a nucleotide chain but not start one
How is the new DNA strand created?
Free nucleotides align opposite their complementary base partners (A = T ; G = C)
What is the role of DNA Polymerase III?
DNA pol III attaches to the 3’-end of the primer and covalently joins the free nucleotides together in a 5’ → 3’ direction
In what ways does DNA Polymerase III move?
As DNA strands are antiparallel, DNA pol III moves in opposite directions on the two strands
How does DNA P III work on the leading strand 3’ to 5’?
On the leading strand, DNA pol III is moving towards the replication fork and can synthesise continuously
How does DNA P III work on the lagging strand 5’ to 3’?
On the lagging strand, DNA pol III is moving away from the replication fork and synthesises in pieces (Okazaki fragments)
What is the role of DNA polymerase I?
DNA pol I removes the RNA primers from the lagging strand and replaces them with DNA nucleotides
Which strand has more RNA primers?
As the lagging strand is synthesised in a series of short fragments, it has multiple RNA primers along its length
What is the role of DNA ligase?
DNA ligase joins the Okazaki fragments together to form a continuous strand
How does DNA ligase join the Okazaki fragments?
It does this by covalently joining the sugar-phosphate backbones together with a phosphodiester bond
Can DNA Polymerase simply initiate replication?
NO
DNA polymerase cannot initiate replication, it can only add new nucleotides to an existing strand
What must first happen for DNA replication to occur?
For DNA replication to occur, an RNA primer must first be synthesised to provide an attachment point for DNA polymerase
What does DNA polymerase do to initiate replication?
DNA polymerase adds nucleotides to the 3’ end of a primer, extending the new chain in a 5’ → 3’ direction
In what form do free nucleotides exist in?
Free nucleotides exist as deoxynucleoside triphosphates (dNTPs) – they have 3 phosphate groups
How does DNA polymerase create the chain?
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
Why does DNA polymerase move in different directions?
Because double-stranded DNA is antiparallel, DNA polymerase must move in opposite directions on the two strands
In what direction does DNA polymerase move on the leading strand?
On the leading strand, DNA polymerase is moving towards the replication fork and so can copy continuously
In what direction does DNA polymerase move on the lagging strand?
On the lagging strand, DNA polymerase is moving away from the replication fork, meaning copying is discontinuous
Why is the lagging strand discontinuous?
As DNA polymerase is moving away from helicase, it must constantly return to copy newly separated stretches of DNA
What is the lagging strand built up in?
This means the lagging strand is copied as a series of short fragments (Okazaki fragments), each preceded by a primer
What must be done before the Okazaki fragments can be joined?
The primers are replaced with DNA bases and the fragments joined together by a combination of DNA pol I and DNA ligase
What is DNA sequencing?
DNA sequencing refers to the process by which the base order of a nucleotide sequence is elucidated
What is the most widely used method for DNA sequencing?
The most widely used method for DNA sequencing involves the use of chain-terminating dideoxynucleotides
How are dideoxynucleotides unique?
Dideoxynucleotides (ddNTPs) lack the 3’-hydroxyl group necessary for forming a phosphodiester bond (structure)
How do dideoxynucleotides affect the DNA chain?
Consequently, ddNTPs prevent further elongation of a nucleotide chain and effectively terminate replication
What is the point of using dideoxynucleotides?
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
What method is used to determine the DNA sequence using dideoxynucleotides?
Dideoxynucleotides can be used to determine DNA sequence using the Sanger method
What is set up? Sanger method 1
Four PCR mixes are set up, each containing stocks of normal nucleotides plus one dideoxynucleotide (ddA, ddT, ddC or ddG)
- Why are PSR mixes used? sanger method
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
- How are the fragments seperated? Sanger method
When the fragments are separated using gel electrophoresis, the base sequence can be determined by ordering fragments according to length
- What can be done to the fragments to make detection easier?
If a distinct radioactive or fluorescently labelled primer is included in each mix, the fragments can be detected by automated sequencing machines
What will happen if the sanger method is conducted on the coding strand?
If the Sanger method is conducted on the coding strand (non-template strand), the resulting sequence elucidated will be identical to the template strand
What is the vast majority of human genome composed of?
The vast majority of the human genome is comprised of non-coding DNA (genes only account for ~ 1.5% of the total sequence)
What are the 5 types of noncoding DNA?
satellite DNA Telomeres Introns Non-coding RNA genes Gene regulatory sequences
STING
What are 3 characteristics of satellite DNA?
tandemly repeating sequences of DNA
structural component of heterochromatin and centromeres
commonly used for DNA Profiling
What are 2 characteristics of telomeres?
role and structure
regions of repetitive DNA at the end of a chromosome
Protects against chromosomal deterioration during replication
What are 2 characteristics of introns?
role and structure
non-coding sequences within genes
are removed by RNA splicing prior to the formation of mRNA
What are 2 characteristics of non-coding RNA Genes?
role and structure
codes for RNA molecules that are not translated into protein
e.g genes for tRNA
What are 2 characteristics of gene regulatory sequences?
role and structure
sequences that are involve din the process of transcription
includes promoters, enhancers and silencers
What is DNA profiling?
DNA profiling is a technique by which individuals can be identified and compared via their respective DNA profiles
What is identified in DNA profiling?
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)
How can tandem repeats be excised? DNA profiling
Tandem repeats can be excised using restriction enzymes and then separated with gel electrophoresis for comparison
Why are DNA profiles unique?
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 larger fragments, while shorter repeats will generate smaller fragments
How is DNA packaged in eukaryotic organisms?
In eukaryotic organisms, the DNA is packaged with histone proteins to create a compacted structure called a nucleosome
What is the role of nucleosomes?
Nucleosomes help to supercoil the DNA, resulting in a greatly compacted structure that allows for more efficient storage
What is the role of supercoiling?
Supercoiling helps to protect the DNA from damage and also allows chromosomes to be mobile during mitosis and meiosis
What is the DNA complexed with and what does it form?
The DNA is complexed with eight histone proteins (an octamer) to form a complex called a nucleosome
What are nucleosomes linked by and what do they form?
Nucleosomes are linked by an additional histone protein (H1 histone) to form a string of chromatosomes
What does a string of chromatosomes form?
These then coil to form a solenoid structure (~6 chromatosomes per turn) which is condensed to form a 30 nm fibre
What is done to the fibres and what do they form? organisation of eukaryotic dna
These fibres then form loops, which are compressed and folded around a protein scaffold to form chromatin
What is the final step of the organisation of eukaryotic DNA, involving chromatin?
Chromatin will then supercoil during cell division to form chromosomes that are visible (when stained) under microscope
What is the structure of a nucleosome?
A nucleosome consists of a molecule of DNA wrapped around a core of eight histone proteins (an octamer)
How are the charges arranged in a histone?
The negatively charged DNA associates with positively charged amino acids on the surface of the histone proteins
What extrudes from the histone?
The histone proteins have N-terminal tails which extrude outwards from the nucleosome
What happens during chromosomal condensation?
During chromosomal condensation, tails from adjacent histone octamers link up and draw the nucleosomes closer together
