7.1 DNA structure and replication Flashcards
What did Hershey and Chase try to prove?
That DNA was the genetic material
What did Hershey and Chase do to the viruses?
They were grown in one of two isotopic mediums in order to radioactively label a specific viral component
What did viruses grown in radioactive sulfur have?
Radiolabelled proteins
Where is sulfur present?
In proteins but not DNA
What did viruses grown in phosphorus have?
Radiolabelled DNA
Where is phosphorus present?
In DNA but not proteins
Once labelled, what were the viruses allowed to do?
Infect a bacterium
What happened once the viruses infected a bacterium?
The virus and bacteria were separated via centrifugation
In hershey and chase, what did the larger bacteria form?
A solid pellet
When were the bacterial pellet found to be radioactive?
When infected with the radioactive phosphorus
When were the bacterial pellet not found to be radioactive?
When infected with the radioactive sulfur
What did the Hershey and Chase experiment demonstrate and how?
That DNA, not protein, was the genetic material because DNA was transferred to the bacteria
In hershey and chase, how did the smaller viruses remain?
In the supernatant
How did rosalind franklin investigate the structure of DNA?
Using a method of X-ray diffraction
What inferences could be made about DNAs structure from the scattering pattern?
-Dna is a double stranded molecule
-Nitrogenous bases are packed together on the inside and phosphates form a backbone
- The DNA molecule twists at regular intervals to form a helix
What forms the outer backbone of DNA?
Phosphates and sugars
What are packaged within the interior of DNA?
Nitrogenous bases
What is DNA composed of an equal number of?
Purines and pyrimidines
What are purines?
A and G
What are pyrimidines?
C and T
In order fo pairing between purines and pyrimidines to occur what must happen?
The two strands must run in antiparallel directions
How many hydrogen bonds are between adenine and thymine?
Two
How many hydrogen bonds are between guanine and cytosine?
Three
What two mechanisms does DNA structure suggest for DNA replication?
Replication occurs via complementary base pairing
Replication is bi-directional due to the antiparallel nature of the strands
What is DNA replication?
A semi-conservative process that is carried out by a complex system of enzymes
What seven enzymes are used in DNA replication?
Helicase
DNA gyrase
SSB proteins
DNA primase
DNA polymerase I
DNA polymerase III
DNA ligase
What does helicase do?
Unwinds and separates the double stranded DNA
How does helicase unwind and separate the DNA?
By breaking the hydrogen bonds between base pairs
Where does helicase do its job?
At specific regions
What does helicase end up creating?
A replication fork of two strands running in antiparallel directions
What does DNA gyrase do?
Reduces the torsional strain created by the unwinding of DNA by helicase
How does DNA gyrase reduce torsional strain?
By relaxing positive supercoils via negative supercoiling that would otherwise form during the unwinding of DNA
What does SSB stand for?
Single stranded binding proteins
What does SSB proteins bind to?
The DNA strands after they have been separated
What two things do SSB proteins prevent?
The strands from re-annealing and the single stranded DNA from being digested by nucleases
What happens to SSB proteins once they’ve done their job?
They will be dislodged from the strand
When will SSB proteins be dislodged from the strand?
When a new complementary strand is synthesised by DNA polymerase III
What does DNA primase do?
Generates a short RNA primer on each of the template strands
What does the RNA primer provide?
An initiation point for DNA polymerase III
What can DNA polymerase III do to a nucleotide chain?
Extend but not start one
Where do free nucleotides align?
Opposite their complementary base partners
Where does DNA pol III attach?
To the 3’ end of the primer
What does DNA pol III do?
Covalently joins the free nucleotides together in a 5’ - 3’ direction
Which way does DNA pol III move on the leading strand?
Towards the replication fork
Which way does DNA pol III move on the lagging strand?
Moving away from the replication fork
How does DNA pol III synthesize on the leading strand?
Continuously
How does DNA pol III synthesize on the lagging strand?
In pieces (okazaki fragments)
Due to the fact the lagging strand is synthesised in a series of short fragments what does it have along its length?
Multiple RNA primers
What does DNA pol I do?
Removes the RNA primers from the lagging strand and replaces them with DNA nucleotides
What does DNA ligase do?
Joins the okazaki fragments together to form a continuous strand
How does DNA ligase join okkazaki fragments?
By covalently joining the sugar phosphate backbones together with a phosphodiester bond
What can DNA polymerase no do?
Initiate replication
What must happen for DNA replication to occur?
An RNA primer must first be synthesised to provide an attachment point for DNA polymerase
How do free nucleotides exist as?
Deoxynucleoside triphosphates
What does DNA polymerase use the energy obtained from cleaving the two additional phosphates for?
To form a phosphodiester bond with the 3’ end of a nucleotide chain
Why must DNA polymerase moved in opposite directions on the two strands?
Because double stranded DNA is antiparallel
What must DNA polymerase do as it moves away from helicase?
Constantly return to copy newly separated stretches of DNA
What are short fragments of copied DNA called?
Okazaki fragments
What precedes each Okazaki fragment?
A primer
What are primers replaced by?
DNA bases
What combination joins fragments together?
DNA pol I and DNA ligase
What is DNA sequencing?
The process of how the base order of a nucleotide sequence is elucidated
What does the most widely used method for DNA sequencing involve?
The use of chain terminating dedddeoxynucleotides
What do dideoxynucleotides lack?
The 3’ hydroxyl group needed to form a phosphodiester bond
What do ddNTPs do?
Prevent the elongation of a nucleotide chain and terminate replication
When using a ddNTP, what does the resulting length of a DNA sequence reflect ?
The nucleotide position where the dddNTP was incorporated
What is the name of the method where dideoxynucleotides can be used to determine DNA sequence?
The Sanger method
What is the result if the Sanger method is conducted on the coding strand?
The resulting sequence will be identical to the template strand
As a typical PCR will generate over 1 billion DNA molecules, what should each PCR mix generate?
All the possible terminating fragments for that particular base
What is the Sanger method?
Where four PCR mixes are set up, each containing stocks of normal nucleotides plus one dideoxynucleotides
When the fragments are separated using gel electrophoresis, what can be determined and how?
The base sequence by ordering fragments according to length
What happens in the Sanger method if a distinct radioactive or fluorescently labelled primer is included in each mix?
The fragments can be detected by automated sequencing machines
What is the vast majority of the human genome comprised of?
Non-coding DNA
What are the five different types of non-coding DNA?
Satellite DNA
Telomeres
Introns
Non-coding RNA genes
Gene regulatory sequences
What is satellite DNA?
Tandemly repeating sequences of DNA which is a structural component of hétérochromosome and centromeres
What is satellite DNA commonly used for?
DNA profiling
What are telomeres?
Regions of repetitive DNA a the end of a chromosome
What do telomeres protect against?
Chromosomal deterioration during replication
What are introns?
Non-coding sequences within genes
What removes introns and when?
RNA splicing prior to the formation of mRNA
What are non-coding RNA genes?
Codes for RNA molecules that are not translated into protein
What are gene regulatory sequences?
Sequences that are involve in the process of transcription
What is DNA profiling?
A technique where individuals can be identified and compared via their respective DNA profiles
What is within the non-coding regions of an individual genome?
Satellite DNA
What is within the non-coding regions of an individual genome?
Satellite DNA
What are the repeating elements of long stretches of DNA called?
Short tandem repeats
What are the repeating elements of long stretches of DNA called?
Short tandem repeats
How can tandem repeats be excised?
Using restriction enzymes
How can tandem repeats be separated for comparison?
Gel electrophoresis
Why will individual have unique DNA profiles?
As they will likely have different numbers of repeats at a given satellite DNA locus
What will longer repeats generate?
Larger fragments
What will shorter repeats generate?
Smaller fragments
What is a Nucleosomes?
The structure of DNA packaged with histone proteins
What is a Nucleosomes?
The structure of DNA packaged with histone proteins
What do Nucleosomes help to do?
Supercool the DNA
What do Nucleosomes help to do?
Supercoil the DNA
What does Nucleosomes supercoiling the DNA result in?
A greatly compacted structure that allows for more efficient storage
What does supercoiling help protect DNA from?
Damage
What does supercoiling help protect DNA from?
Damage
What does supercoiling allow chromosomes to do?
Be mobile during mitosis and meiosis
How are Nucleosomes are linked?
By an additional histone protein
How are Nucleosomes are linked?
By an additional histone protein
What is linked Nucleosomes called?
Chromatismes
What are linked Nucleosomes called?
Chromatosomes
What do chromatosomes coil to form?
A solenoid structure
What do solenoid structures condense to form?
30 nm fibre
What do the 30nm fibre form?
Loops which are compressed and folded around a protein scaffold to form chromatin
What do the 30nm fibre form?
Loops which are compressed and folded around a protein scaffold to form chromatin
What does chromatin do to form what?
Supercoil during cell division to form chromosomes
What is eight histone proteins called?
An octamer
What does the negatively charged DNA associate with on the surface of the histone proteins?
Positively charged amino acids
What do histone proteins have?
N-terminal tails
What do n-terminal tails on histone proteins do?
Extrude outwards from the Nucleosomes
What do tails and octamers do during chromosomal condensation?
Link up and draw the Nucleosomes closer together
