What are nucleotides?
nucleotides join up to make nucleic acids
- 5 prime end joins to the 3 prime end and vice versa
- forms DNA or RNA depending on the sugar
are made up of
- a nitrogenous base = A, T, C, G or U
- a sugar = ribose (RNA) or deoxyribose (DNA)
- a phosphate molecule
How is DNA structured/arranged?
arranged in a double helix
- helix is right handed
- distance occupied by one complete turn of the helix is 34A which is equal to the distance of 10 base pairs
- helix is not even = has major and minor grooves
- strands are held together by interactions between bases = hydrogen bonds
purines (A,G) interact with pyrimidines (C, T and U)
What are the advantages of DNA structure/arrangement?
structure is stable
- bases on the inside are protected
- has high mechanical strength
easy to copy the strands
- template is always present
What are the different forms of DNA?
A form
- shorter than the B form (initially described) = 28A
- right handed
- thicker
- are RNA-DNA or RNA-RNA hybrids
B form
- 34A distance
- right handed
Z form
- longer than A and B forms
- left handed
- thinner
How can DNA be packaged to reduce its size/length?
supercoiling
packaging in proteins
What are topoisomerases?
enzymes which cause changes in the degree of coiling
- introduce or eliminate coiling
topoisomerase II
- DNA gyrate = introduces supercoiling
topoisomerase I
- eliminates supercoiling
What is the difference in features of overwound and underwound DNA?
overwound = positive supercoiling
- involves twisting towards the helical conformation = towards the direction the helix is already coiled/twisted
- helix begins to distort and knot
- increase in linkages on the helix
underwound = negative supercoiling
- involves twisting against the helical conformation/direction
- unwinds and straightens the helix
- further helical stress can be removed by partial strand separation = hydrogen bonds break and part of the strand separates
How do organisms store their DNA?
DNA is stored in the negatively supercoiled form
- decreases storage space
- allows for easier opening of the helix = promotes DNA replication and transcription
How does topoisomerase I work?
cannot introduce supercoiling = does not use ATP
stimulates relaxation of the supercoiled DNA
- reduces twists in the DNA strand
- does not require ATP to function
cuts a single strand of the double helix and passes the other strand through it. it reseals and the helix forms with one less twist
How does topoisomerase II work?
introduces supercoiling
- can introduce positive or negative supercoiling
- requires ATP
cuts both strands of the double helix and passes a second DNA duplex (double strand) through the break then reseals the break
- can increase or reduce the linkage number by 2 units at a time
examples - DNA gyrase
How can DNA be packaged into proteins?
prokaryotes
has circular DNA
- has superhelical domains that are separated by RNA or protein ‘locks’
- DNA is highly condensed
- is stored in a region of space called the nucleoid - fibrous structure
nuceloid
- region where DNA transcription and replication take place
- contains a combination of proteins
proteins bind to DNA and alter its shape and ability to replicate, recombine, repair
- proteins including Hu, IHF, Fis
How can DNA be packaged into proteins?
eukaryotes
has linear DNA
DNA must be compacted into nucleosomes to fit into the cell nucleus
nucleosomes
- segments of DNA wrapped around histone octamers (8 histones)
- are the smallest units of chromatin = chromatin condenses to form chromosomes
What are the types of chromatin?
euchromatin
- are open and active
- involved in active transcription of DNA to mRNA
- stains lightly
heterochromatin
- are closed and mainly silent = more tightly packaged and highly condensed so is not transcribed
- stains darkly
How are nucleosomes formed?
formed DNA being associated with proteins = histones
histone octamer
- is a 8 protein complex
- contains two copies each of the histone proteins = H2a, H2b, H3 and H4
DNA wraps around the histone octamer to form nucleosomes
- are held together by H1 which is a linker protein = binds at the entry and exit regions of DNA, sits at the junction between nucleosomes and completes/holds together the nucleosome
What are the different types of DNA replication? What is the form that occurs in us? How can it be proven?
dispersive replication
- first and second generation daughter strands contain some of the new and old strand
conservative replication
- first generation has one completely new and old strand
- second generation has 1 old strand and 3 new strands
semi-conservative replication
- first generation = each daughter cell has a single old and new strand
- second generation = has two new daughter strands and 2 made of the old and new strands
Meselson Stahl experiment
- uses isotopes of nitrogen (14 and 15) to determine how the strands replicate
What is the process of DNA replication?
initiation
- DNA is pulled apart at the origin = site of replication and is a fixed point in eukaryotes and prokaryotes
- origin is recognised by proteins
- direction of movement can be unidirectional (both strands replicate in the same direction) or bidirectional (strands replicate in opposite directions)
elongation
- catalytic reaction on the DNA polymerase which can only synthesis in the 5 to 3 prime direction
termination
- RNA primers are removed by RNase H enzyme
How does the elongation section of replication occur?
DNA helicase unwinds the double stranded helix
- begins at the origin and breaks the hydrogen bonds
- forms the replication fork in the strands
DNA primase synthesises RNA primer to lay down a short stretch of 3 prime RNA for DNA polymerase to add on nucleotides
- in the leading strand DNA polymerase then moves and begins to add on nucleotides continuously = 5 to 3
- in the lagging strand DNA polymerase attaches the nucleotides to the sections available
- as DNA helicase opens more of the DNA strand and the replication fork progresses the DNA polymerase must constantly return to copy newly separated stretches of DNA = creates fragments
- at each new fragment, a RNA primer must precede it to initiate DNA polymerase
- removal of the primer by RNase H leaves gaps/nicks in the strands which must be fused together by DNA ligase
Why is replication semi-discontinuous?
two strands of DNA are being replicated in opposite directions
- one strand is going from 5 prime to 3 prime while the other is going from 3 prime to 5 prime
leading strand
- goes from 5 prime to 3 prime = forms continuously
lagging strand
- does from 3 prime to 5 prime = forms discontinuously as DNA polymerase falls off synthesis the newly separated strands then the pieces are fused together
What is the role of in DNA replication? DNA helicase DNA polymerase primase RNA primer DNA ligase topoisomerase single strand binding proteins
DNA helicase
- unzips/unwinds the DNA double helix by breaking the hydrogen bonds between bases
DNA polymerase
- joins nucleotides together to form a new strand
- adds the nucleotides onto the 3 prime end of the primer
Primase
- forms the RNA primer which initiates DNA polymerase
RNA primer
- form provides an attachment point for DNA polymerase which can only bond to 3 prime OH
DNA ligase
- joins/fuses together the okasaki fragments
Topoisomerase
- works at the region ahead of the replication fork to prevent DNA from being overwound (positive supercoiling) = can be overwound if DNA helicase pulls apart DNA which is fixed
Single strand binding proteins
- protect single strand DNA during generation
- stops it from being attacked by nucleases and prevents rewinding of DNA at the replication fork
How is RNA primer removed from the newly synthesised strands?
can be removed by RNase H
- enzyme
- leaves nicks in the strands
can be removed by 5 prime to 3 prime exonucleases
- DNA polymerase I can be used to remove primers
What is the processivity of DNA polymerase?
describes the polymerases activity
- describes the number of bases that DNA polymerase can synthesis in a single association with the template = without being released
processive polymerisation
- can synthesis a large number of bases = suitable for DNA replication = example is DNA polymerase III
distributive polymerisation
- can only synthesise a few bases at a time = suitable for DNA repair = example is DNA polymerase I
What is the difference between 5 to 3 prime exonuclease activities and 3 to 5 prime exonuclease activities?
5 to 3 prime exonuclease
- can remove primer
- polymerase activity can fill nicks/gaps
3 to 5 prime exonuclease
- used in proof reading or editing the DNA
- can remove mismatched nucleotides/bases
How can DNA be damaged endogenously and exogenously?
endogenous damage - inside the cell
- replicative errors = incorrect bases, insertion, deletion
- oxidative damage by free radicals
- spontaneous alteration
- alkylating agents
exogenous damage - damage from outside
- UV
- pollution
- carcinogens
- radiotherapy
How are error in DNA dealt with?
direct reversal
- damaged area is repaired directly, does not require nucleotide template
nucleotide excision repair (NER)
- error is removed as a stretch of nucleotides
- typically done by UV damage
base excision repair (BER)
- only the affected base is removed
mismatch repair
- mismatched bases = similar to NER
- removes whole stretch of nucleotides
recombination repair
- repairs double strand breaks
- uses sequence from homologous pieces of DNA = occurs during S phase
non-homologous end joining (NHEJ)
- joins ends of DNA even if they are not related/homologous
- proteins brings the ends together then DNA ligase joins them
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