7.1 DNA replication Flashcards
What was the Hershey-Chase experiment?
- Virus (T2 bacteriophage) were grow in one of the two isotopi cmediums in order to radioactively label a specific viral component - radioactive sulfuer (³⁵S) had radiolabelled proteins present in proteins not DNA, radioactive phosphorus (³²P) had radiolabelled DNA (phosphorus is present in DNA not proteins)
- Viruses alloed to infect E coli
- Virus and bacteria were separated via centrifugation
- larger bacteria formed a solid pellet
- smaller virus remain in the supernatant
- bacteria pellet found to be radioactive
- DNA, not protein was the genetic materialbecause DNA was transferred to the bacteria but the protein was not
How did Rosalind Franklin contribute to the DNA helical structure?
provided X-ray diffraction
What is X-ray diffraction?
- purified and crystalised DNA is targetted by an x ray beam
- x ray beam diffract when they contact an atom
- scattering pattern is recorded on a film and used to identify molecular structure
What deductions can be make?
- the cross in the centre of the pattern indicated that the molecule was helical in shape
- the angle of the cross shape showed the pitch (steepness of angle) of the helix
- the distance between the horizontal bars showed turns of the helix to be 3.4 nm apart
- the distance between the middle of the diffraction pattern and the top showed that there was a repeating structure within the molecule, with a distance of 0.34nm between the repeats. This turned out to be the vertical distance between adjacent base pairs in the helix
Where did Watson and Crick collated evidence from?
- Linus Pauling - pioneered molecular modelling
- Franklin’s X-ray diffraction experiments - demonstrated that phosphates and sugars form an outer backbone and nitrogenous bases are packaged within the interior
- Chargaff complementary base pairing - demonstrated that DNA is composed of an equal number of purines (A+G) and pyrimidines (C+T) and in order for the pariing between purines and pyrimidines to occur, the two strands must run in antiparallel directions
- Their own findings - adenine and thymine paired via two hydrogen bonds, whereas guanine and cytosine paired via 3 hydrogen bonds, A-T bond is the same length as G-C and the complementary nucletides bonded ‘upside down’ in relation to one another
What is one difference between eukaryotic and bacterial DNA?
eukaryotic DNA is associated with proteins called histones
prokaryotes are not so they are referred to as being naked
What are histones used by the cell to do?
package DNA into structures called nucleosomes
What does a nucleosome consist of?
a central core of 8 histone proteins (octamer) with DNA coiled around the proteins
What does an octamer consist of?
two copies of 4 types ofhistones
How are nucleosomes connected?
By linker DNA
What is H1 and what does it do?
its an additional histone protein molecule that serves to bind the DNA to the core particle
What is sueprcoiling due to?
the association of histones with the DNA contributes to this pattern
What does supercoiling allow?
allow great length of DNA to be packed into a much smaller space within the nucleus
What is a nuecleosome?
an adaption that facilitates the packing of the large genomes that eukaryotes possess
What do H1 histones bind to form?
form a structure called the 30nm fibre that facilitates further packing
How does synthesis on the two strand occur?
in very different ways due to the anti-parallel fashion
How is DNA made on the leading strand?
it is made continuously following the fork as it opens
How does the lagging strand replicate DNA?
is made in frgments (Okazaki fragments) moving away from the replication fork
new fragments are created on the lagging strand as the replication fork exposes more of the template stand
What proteins are involve in DNA replicaiton?
enzymes
What is the process of DNA replication?
- enzyme helicase unwinds DNA at the replication fork
- enzyme topoisomerase releases the strain that develops ahead of the helicase
- single-stranded binding proteins keep the strands apart long enough to allow the template strand to be copied
- the enzyme DNA primase creates one RNA primer on the leading strand and many RNA primers on the laggin strand
- DNA polymerase III reads the parent template strand in a 3’C to 5’C direction and builds the leading strand in the 5’C to 3’C direction
- free phosphorylated nucleotides are bonded to the exposed bases, following complementary base pairing rules and joined by condensation reaction catalysed by DNA polymerase III (also proof reads)
- Lagging strand is also build in a 5’C to 3’C direction, away fromthe replication fork
- it is build discontinuously in Okazaki fragments
- DNA ligase join the okazaki fragments
- DNA polymerase I removes the DNA primers from the newly synthesised strands and replaces them with DNA nucleotides
Within DNA molecules, where do DNA replicaiton begin?
at sites called origin of replication
How many orogins of replication are there in eukaryotes and prokaryotes?
many in eukarotes
just one in prokaryotes
Which directions does replication in prokaryotes occur?
in both directions away from the origin
this result appears as a replication bubble in electorn micrographs
Where is the phosphate group of new DNA nucleotides added? What does it say about the direction in which replicatio occurs?
to the C’ carbon of the deoxyribose of the nucleotide at the end of the chain
replication therefore occurs in the 5’ to 3’ direction
What are coding sequences?
DNA sequences that code for the production of polypeptides
What are some function of non-coding sequences found in genomes?
- produce tRNA and rRNA
- regulation of gene expression such as enhancers and silencers
- introns
What are the two types of repetitive sequences?
Moderately repetitive sequences and highly repeptitive sequences (satellite DNA)
Where are repetitive sequences ?
on the ends of eukaryotic chromosomes called telomeres
What are the function of telomeres? How?
serves a protective function
during interphas,e the enzymes that replicate DNA cannot continue replication all the way to the end of the chromosome
if cells went through the cell cycle without telomeres, they would lose the genes at the end of chromosomes
Sacrificing the repetitive sequences found in telomeres serves a portective function
What is a variable number tandem repeat? (VNTR)
a short nucletide sequence that shows variations between individuals in terms of the number of times the sequence is repeated
each variety can be inherited as an allele
What is a locus?
a physical location of a heritable element on the chromome
How do genealogists deduce paternal lineage?
by analysisng short tandem repeats fro the Y-chromosome, and deduce maternal lineage by analysing mitochondrial DNA variations in single nucleotides at specific locations called hyper-variable regions
What happens in DNA sequencing?
- Many copies of the unknown DNA that is to be sequences are placed into test tubes with all of the raw materials including deoxyribonucleotides and the enzymes necessary to carry out replication
- very small quantities of dideoxyribonucleotides that have been labelled with different fluorescent markers are added
- the dideoxyribonucleotides will be incorporated into some of the new DNA
- when they are incorporated, they will stop the replication at precisely the point where they were added
- the fragments are separated by legnth using electrophresis
- the sequence of bases can be automatically analysed by comparing the colour of the fluorescence with the length of the fragment
