DNA Flashcards
How do you prove that DNA is the genetic material
Take a pathogenic bacterium (S strain) which undergoes a random mutation forming R strain (non-pathogenic).
Grow R cells in presence of heat killed S cells
R strain cells are transformed to S strain whose daughters are pathogenic
Test each molecule for transformation: only DNA results in S strain
What do S strain cells cause
Pneumonia
3 things DNA needs to do
Be stored
Be propagated
Be read
What are nucleic acids made of
Sugar
Phosphate
Aromatic Bases
How remember purine from Pyrimidine
Purine: 2 rings
Pyrimidine: 1 rings
Which bases are purines and Pyrimidines
Purine:
A
G
Pyrimidine
T
C
U
Is DNA branched
No it is a linear chain of nucleotides
5’ to 3’ polarity
Which is the 5’ end
Where phosphate bonds to CH2
Which is the 3’ end
Where phosphate group binds directly to C in ring
Give 6 types of nucleic acid polymers
DNA mRNA tRNA rRNA miRNA IncRNA
What is chargaff’s rule
%A=%T
%C=%G
What does it mean to say DNA is an anti parallel double helix
One strand is 3’ to 5’ and the other is 5’ to 3’
How many base pairs per turn
Why is it not an integer
10.5
It is an average
How are the planes of bases related to the helix axis
Perpendicular
What basic fact about sequence specific recognition by transcription factors is important
The edges of bases are exposed to solvent
Give and briefly describe the 3 types of DNA
Which is most common
A-DNA - squashed, right handed
B-DNA - most common
Z- DNA - left handed, less regular, less stable and therefore transient
How much DNA must be packed into a cell?
What is the average cell size?
How is this done
2m
10μm
Supercoiling
What is supercoiling
The coiling of a coil
In DNA it’s when the axis of the double helix is wound upon itself
Is the supercoiled state relaxed
No it is high energy and generates structural strain
How is DNA usually wound
Why
Underwound
Facilitates compaction and strand separation
How can supercoiling be measured
Topology
How can supercoiling be controlled
Enzymes can change the degree of cooling. These are called topoisomerases
How do topoisomerases work briefly
Cleaning and rejoining DNA strands
What is a nucleoid
A rosette model of DNA organisation
What is the basic unit of eukaryote chromosome structure
The nucleosome
What is a nucleosome
where DNA is wrapped around a protein barrel made of a Histone octamer
DNA wraps 2x around the barrel
Are histones charged
Yes they are positive
What histones do nucleosomes contain
How much DNA is there
2 copies of H2A, H2B, H3 and H4 and one copy of H1
200 nucleotides worth
What does H1 do
It is a linker histone, linking the entry and exit points of DNA as well as binding DNA between nucleosomes
What is a chromatin
Chromosomal DNA packaged with histones. It’s simplest form is the 10nm fibre
What is a euchromatin
An open chromatin which is more accessible (thus transcriptionally active)
Like beads on a string
What is a heterochromatin
Condensed chromatin Less accessible (therefore transcriptionally inactive)
Known as a solenoid
What is SMC
Structural. Maintenance of Chromosome proteins
Multi-domain ATPases
Give overview of SMC dimer
2 molecules of anti parallel coil with a head and hinge at either end and an arm in between
Hinge of each join the two molecules and the head is where the ATP binds
Give the 2 important SMC proteins
What do they do
Cohesin
Condensin
Encircle DNA
What are cohesinopathies
Developmental disorders caused by mutations in Cohesin leading to defects such as limb deformities and craniofacial anomalies
What is the loop extrusion model
Condensin extrude loops of DNA
Condensin molecules approach each other and arrange around the longitudinal axis to form threadlike structures
These structures pack in layers to form an X shaped chromosome
Give 4 implications of DNA packing
Chromatin status
Accessibility for gene transcription
Local chromatin status in dynamic
Alterations in chromatin status are important in disease
What is chromatin status
The degree of DNA compaction that regulates its accessibility
3 things which affect chromatin status
H1: higher H1 levels favour chromatin condensation (less accessible)
Post translational modifications if histones that reduce positive charge (lysine acetylation)
Chromatin remodelling
How are histones modified
Covalently by post translational modifications on the unstructured tails that project out eg methylation or phosphorylation
These can be inherited (epigenetics)
What does lysine acetylation do to histones
Reduces compaction by removing positive charge having a direct effect on chromatin condensation
Are histone modifications static?
No they are dynamic: chromatin writers and erasers add and remove chemical signals
What do chromatin readers do
Recognise each unique set of modifications (Histone Code) and trigger a transcriptional response
What are Bromodomain proteins
A family of chromatin readers that are used to treat cancer as they recognise acetylated Lys
Why is the nucleosome position important?
Are they randomly placed and even?
It affects availability of DNA binding sites for transcription factors
No: there are regions of high nucleosome density and nucleosome free regions
What is nucleosome remodelling
The assembly, movement and editing of nucleosomes, which requires ATP hydrolysis
What are nucleosome remodellers
Protein complexes with ATPase activity
They all have a DNA translocation motor and reader subunits for targeting the remodeller to specific chromatin sites
What does nucleosome assembly involve
A strand of DNA undergoes deposition of H3-H4 tetramer to give random spacing.
Then the nucleosomes are matured and spaced regularly by enzymes
How may a nucleosome remodeller change chromatin access
Repositioning to give irregular spacing
Nucleosome ejection
Histone dimer eviction
What does INO80 do
Nucleosome editing By exchanging histones
Are interphase chromosomes arranged randomly in the nucleus
No they occupy spatially distinct regions called chromosome territories
WHat is Burkitt’s lymphoma
Translocation between MYC Gene and one of 3 immunoglobulin Gene variants located on different chromosomes
What is the most common translocation in Burkitt’s lymphoma
What is the order of likelihood and why
MYC:IGH
IGH>IGL>IGK
IGH is spatially the closest to MYC
What is a TAD
Topologically Associating Domains
A contiguous region along the chromosome which contains the majority of interaction sites
TADs are made of many chromatin fibres
Order TAD, compartments, territories and chromatin fibres according to size
Territory> compartment> TAD> chromatin fibre
What are the 3 theories of DNA replication
Semi conservative
Conservative
Dispersive
What is semi conservative replication
When DNA splits into 2 strands which are transcribe another strand and are incorporated into it
Describe conservative replication
2 newly synthesised strands form an identical double helix while the original DNA remains as it is
Who identified the mode of DNA replication
Meselson and Stahl, 1958
How did Meselson and Stahl prove DNA replication
E. Coli culture was put into a radioactive NH4Cl medium where the N is N-15. This is transferred to a N-14 NH4Cl medium.
They took a sample after 20 mins (1 cell division) and 40 mins (2 cell divisions)
DNA extracted using density gradient centrifugation
What would have been Meselson and Stahl’s possible outcomes for their experiments
Conservative: 2 bands after centrifuge (1 parent and 1 daughter)
Dispersive: 1 band (both a mix of parent and daughter), 1 band after 2nd division
Semi conservative: 1 band (both half parent half daughter) then 2 bands after 2nd division (1 for just new DNA and 1 for original and new mix)
Describe the initiation of replication in prokaryotes
E. Coli has 1 origin which is defined by initiator proteins
Bacterial initiator is DnaA which binds to oriC
What is the OriC locus made of?
5 DnaA boxes and an A/T rich region
Why is it important that the OriC region is A/T rich?
They have fewer Hydrogen bonds so are weaker and can be untwisted more easily
What does the DnaA do
Occupies DnaA boxes and forms a large protein- DNA complex
DnaA binding facilitates unwinding of AT-rich region
DnaA polymerises onto ssDNA of unwound origin
DnaB helicase is recruited to the unwound origin with the help of loafer protein, DnaC
Which enzyme type unwinds DNA
Helicases
What is the structure of a replicative helicase
Hexameric ring with ATP at the interface between subunits
One DNA strand it threaded through the ring as the other is peeled off
Require ATP hydrolysis
What stops 2 strands sticking back together
SSB: binds to both strands to stop rebonding but is easily hydrolysed otherwise it would interfere and damage signalling
What does DNA ligase do
“Glues” together Okazaki fragments
Which direction does DNA polymerase work
5’ to 3’ direction
What does topoisomerase do
Unravel double helix
What is Chargaff’s rule
%A=%T
%G=%C
What is it called when the 2 strands of DNA come apart
Melting
What did Max Delbrück say
The DNA strands would have to be pulled apart to replicated/ be copied
This is the untwiddling problem
What does DnaA do
Creates a small melted region which acts as a loading site for DnaB
What is DnaB
A helicase that uses ATP hydrolysis to direct unwinding of double stranded DNA
How do replication forks arise
Replication initiates at the origin and spreads bidirectionally giving 2 y-shaped structures
What enzyme is responsible for synthesising DNA
Give 2 difficulties of this enzyme
DNA polymerase
Only works in 5’ to 3’ direction
Can only extend synthesis from a pre-existing 3’ OH group of DNA
How to get over the fact that DNA polymerase cannot begin chain synthesis itself
Primer
Primase, a specialised RNA polymerase that acts to make a short RNA molecule that is then extended by DNA polymerase
Which strand is the leading strand
3’ to 5’
How is the lagging strand replicated
In short pieces called Okazaki fragments which are then stitched together to make a continuous strand
How long are Okazaki fragments in
a) humans?
b) bacteria?
a) 100-200 bases long
b) 1000 bases long
Why must RNA primers be removed
The genome is made of DNA only
What enzyme carries out DNA replication in bacteria
What happens when it encounters an RNA primer
DNA polymerase III
it stalls when it encounters RNA ahead of it and the enzyme DNA polymerase I
Is recruited to the 3’ OH left by DNA polymerase III
How does DNA polymerase I deal with RNA primers
The 5’ to 3’ exonuclease activity chews this up, allowing the polymerase activity to make DNA in its place
What is 5’ to 3’ exonuclease activity
Hydrolyses RNA and DNA from 5’ end
Describe the difference in leading and lagging strand activity in E. coli
Leading: goes half way round the chromosome where it meets the fork coming in the opposite direction (keeps going for 2,500,000) bases
Lagging: only goes for 1000 bases
Discuss the processivity of DNA polymerase
Not great
It can’t hold onto the DNA v well so relies on a sliding clamp (the β clamp) to ensure it remains attached to the strand and is capable of processive synthesis
How many times does the β clamp have to attached to a single strand
Once on leading strand
Many times on lagging strand
What is Nick translation
5’ to 3’ activity by DNA polymerase I
What is the sliding clamp?
It is the β subunit of DNA polymerase III
What is the holoenzyme complex
The interaction of several proteins to form one replication machine
It acts to couple leading and lagging strand synthesis with replication form progression
Why was the trombone model proposed
Leading and lagging strands head off in different directions and must be brought together by holoenzyme complex
How does the trombone model work
The lagging strand is primed then bent back around to engage with DNA polymerase III forming a loop of increasing size that is periodically released allowing a novel priming event to occur
What is the DNA replication sequence
Origin recognition Helicase recruitment DNA melting Priming Elongation Association of polymerase with a sliding clamp
Why do eukaryotic cells have multiple origins of replication
To all DNA replication in a reasonable timeframe
What recognises origins in eukaryotic chromosomes
A 6 protein complex called ORC
What does ORC stand for and what does it do
Origin Recognition Complex
Recruits the DNA melting helicase (MCM, which also has 6 subunits)
What does ORC require to recruit MCM
Cdc6 and Cdt1
What is the most significant mechanistic distinction between prokaryotic and eukaryotic DNA replication
The way in which primers are removed
How are RNA primers removed in eukaryotes
What kind of enzyme is used
By RNaseH which digests RNA that is base paired to DNA. This structure is then cut off by Fen1 and the resulting nick is fixed by DNA ligase
This processes are coordinated by the eukaryotic sliding clamp, PCNA
A specialised ribonuclease
Why is DNA in eukaryotes controlled by the cell cycle
So DNA is replicated only once per cycle
What worries Watson and Crick about their model and replication
Since it is 2 chains intertwined it was difficult to foresee how they would separate and replicate without tangling
What stops replicating DNA tangling
Topoisomerases which release topological tension in DNA molecules
How does each type of topoisomerase work
Type I: nick a single strand and swivel it around another before resealing the nick
Type II: cut both strands and allow another duplex to pass through the gap, which is then resealed
Does DNA topoisomerase 1 require ATP
Why (2)
No
Formation of covalent bond is isoenergetic with the phospho-ester bond
DNA swivels due to the torque caused by supercoiling
Which drugs target the action of topoisomerases
Etoposide and doxorubicin as inhibitors for cancer chemotherapy
What are often exploited in eukaryotes for Drug therapy (DNA replication)
MCMs as markers of cellular proliferation
What is a genome
The complete set of genetic material for that organism (usually DNA expect for some viruses that use RNA)
In eukaryotes what DNA is included in the genome
The DNA found in nuclear chromosomes
Not mitochondrial DNA
Is a chromosome all equally condensed
No there are some less dense regions (euchromatin) and more condensed regions (heterochromatin)
Which regions of the genome are not transcribed
The heterochromatin
What are the 2 major regions of heterochromatin in mammalian chromosomes
Centromeres and telomeres
What are centromeres
Where are they found
Regions of the chromosome that contain repetitive sequences and are essential structures for chromosome pairing and mitosis
In the middle of the chromosome in humans but at one end of mice chromosomes
What are telocentric chromosomes
Centromeres in mice found at the end of the chromosome
Does centromere position matter
Yes as it determines the length of the two arms of the chromosome
Which arm is shorter in a chromosome
p (like petit)
q is the long one
What happens in extreme forms of DNA condensation into heterochromatin
Inactivation of the X chromosome in female (XX) cells to form Barr bodies
What is Lyonisation
Formation of Barr bodies by extreme DNA condensation into heterochromatin
Named after Mary Lyons
It results in cells only having one active copy of the X chromosome
How and why are X chromosomes in females silenced
Women have XX so one is silenced so you don’t have excess copies of all the genes in the X
It is silenced by compacting it into heterochromatin resulting in dosage compensation
Which X is silenced in females
Random
This leads to the patchwork of colours on a calico cat coat
Can male calico cats have different colour patches
Usually no, in theory only female
But there are some rare examples of Male cats who have extra copies of the X chromosome
This is the feline equivalent of Klinefelter’s syndrome
What is Klinefelter’s syndrome
Give symptoms
When males are born with an extra X chromosome
May lead to slower development, dyslexia, difficulty socialising, and in adult life leads to more feminine features as well as infertility and reduced sex drive
Why does the lagging strand replication mechanism make telomeres a requirement
What would happen without telomeres
The end of the chromosome either isn’t replicated or is left with an RNA-DNA duplex
There would be a progressive shortening of chromosome and potential loss of genetic information as the RNA would not be replicated
What is the telomere sequence in humans
What does it do
TTAGGG
Produces a protective cap for the chromosome end
How is a telomere added
Via the action of telomerase
Discuss the structure of telomerase
Contains both a protein and RNA component
The RNA component serves as a template for DNA synthesis making telomerase a self templating reverse transcriptase
Why are reverse transcriptases important
Where are they often found
They use RNA to make DNA (over turning the central dogma proposed by Crick)
In many RNA viruses eg HIV
How does telomere action counteract the lagging strand problem
Adds long repetitive stretches of single stranded DNA to the end of the chromosome
This DNA can adopt some unusual structures such as a loop that closes off the end of the chromosome (the T loop) and quadruplex DNA structures
What is the purpose of quadruplex DNA structures
Prevent the exposed chromosome ends from being mistakenly recognised by the cell’s DNA damage response systems as damaged DNA
Why does telomerase add such a long stretch of DNA to the end of each chromosome
Normal adult differentiated cells have low levels of telomerase so each time they divide, their telomeres get shorter. Once telomeres reach a critical length the cells read this as getting old and stop dividing. Reintroduction of telomeres increase lifespan
What contributes to immortality in cancer
Cancer cells have elevated telomerase levels
How can knowledge of telomeres help cancer treatment
Telomerase inhibitors may work as possible anti cancer drugs
Which DNA sequencing method did Sanger invent
Chain terminator method
What does the Sanger method of DNA sequencing rely on
The fact DNA polymerase can incorporate 2’3’- dideoxynucleotides (ddNTPs) into growing DNA chains
As the ddNTP does not have a 3’ OH, it terminates the chain
What was the original technique used by Sanger
4 reactions set up where DNA synthesis is primed by synthetic oligonucleotides. The dNTPs had a radio label and were spiked with a ddNTP
This meant the new DNA chains randomly terminate at either A G C or T residues
Then perform gel electrophoresis
The gel is exposed to X Ray film to visualise the DNA bands and the sequence is read from bottom to top
How is the Sanger method done now
Uses ddNTPs conjugated with fluorescent markers, enabling a sequencing reaction
Fine capillaries are now used for separating DNA fragments not gels
What is reannealing
The study of DNA composition by studying the rate at which denatured DNA reformed into double stranded DNA in solution
More complex DNA structures take longer to reform
Why does more complex DNA take longer to form
The probability of finding a complementary stretch of DNA in a complex population is lower
How big is an E. coli genome
What about yeast
4.7 x10^6 bases
13x10^6
How big is the human genome
> 3x10^9
How big is the genome of A. Dubia (an amoeba) compared to human
200x larger in the amoeba
Does bigger genome= bigger genes
No
The worm C. elegans has 10^8 bases but 19,000 genes
The human has a genome of 3x10^9 bases but has only 25000
Human genome is 250x bigger than those orb of yeast but only encodes 5x more genes. Why is this
Human coding sequences are stuffed with junk DNA
95% is thought to be non-coding
What are the 4 classes of repetitive sequence found in the human genome
Give their % of the genome
Simple sequence repeat (CACACA) (3% of. Genome)
Segmental duplications (5%)
Transposon derives repeats (45%)
Inactive copies of partially retrotransposed cellular genes
Is the genome static
No
What are mobile genetic elements
Give 2 broad types
Regions of DNA that can move around and insert in other parts of the genome
This can cause mutations depending where they insert
Transposons and retrotransposons
What moves DNA transposons
Transposase
What are retrotransposons
Where DNA is transcribed to RNA and then reverse transcribed to DNA which is Inserted back into a different part of the genome
What are “jumping genes”?
Mobile genetic elements
Why are transposons bad for antibiotics
Many transposons carry antibiotic resistance genes so can propagate resistance within and even between strains and species
Give 2 examples of diseases resulting from the mutagenic quality of transposition
Muscular dystrophy
Haemophilia
How could transposons be useful
Facilitate shuffling of coding sequences or moving genes so that they are under the control of a new promoter (changing when and where it is expressed)
They may help generate our immune system diversity system
What do recombinases do
Mediate diversity generation are related to transposon-encoded enzymes and may therefore be derived from an ancient transposon
Give uses of the genome sequencing in medicine
Genetic diagnosis
Mutation detection
How does genome sequencing help with CF
Can diagnose the type of CF and therefore it’s severity and the patient’s long term survival
What does reannealing of DNA depend on
Temperature
Length of DNA
base composition
Iconic composition of the solvent
What are DNA endonucleases
Enzymes in bacteria that digest DNA to destroy incoming viral DNA
What is the restriction modification system
The anti-viral defence in bacteria that uses DNA endonuclease to destroy incoming viral DNA
How do DNA endonucleases distinguish between bacterial and viral DNA
They recognise specific sequences which may be absent from the bacterial genome
Also specific for methylated or non methylated DNA
Where can DNA be methylated
A or C residues
Why is it called the restriction modification system
Bacteria modify their DNA so they can selectively attack viral DNA, restricting the viral infection
Which type of restriction enzyme are most widely used
Type II
These recognise sequences that are commonly palindromic and can range from a couple of bases to tens of bases
What is a sticky end
When a restriction enzyme cuts each strand in different positions, generating overhanging stretches of DNA
alternatively, the enzyme could simply cut both DNA strands to generate 2 blunt ended DNA molecules
What can DNA ligase and restriction enzymes do together
Pasting together bits of DNA that have compatible sticky ends (making DNA constructs)
Is DNA charged
What other feature is useful
Yes it is negative (due to phosphate backbone)
Chemically stable in solution
As DNA is negative and stable in solution. What does this property allow
DNA can be separated by size when put in an electrical field in a porous material (usually an agarose or polyacrylamide gel)
After separation DNA can be visualised directly by dyes that bind double stranded DNA and fluorescence under ultra violet light
What is a common dye used for DNA
Ethidium bromide which intercalates into DNA
However it is a mutagen and probs a carcinogen
What is the southern blot
Separate DNA in an electrical field with fluorescent dye
Transfer DNA from the gel to a filter
Analyse using hybridisation to check for specific sequences
What is the most common way to copy your favourite piece of DNA
Cut and paste it into a plasmid And introduce it into the appropriate bacterium
Now your DNA will be copied and amplified and easily purified back from growing bacteria
What is a plasmid
Circular pieces of extrachromosomal DNA in bacteria
How do you copy and paste your favourite DNA
Use restriction enzymes to cut the plasmid open so you can ligase your piece of DNA into the circle
What is an alternative to plasmids for DNA copying
Using bacteriophages
Use restriction enzymes and DNA ligase to cut along with the virus when the virus infects the bacteria
What is a disadvantage of using plasmids or bacteriophages to copy DNA
There is a limit to the length of DNA you can propagate
What is PCR
Polymerase chain reaction
How does PCR work
You know the sequence at the end of the stretch of DNA so you design short primers which provide a free 3’ end of DNA that DNA polymerase can extend from
It proceeds in a series of cycles, increasing the amount of DNA exponentially
Give the 3 steps in each cycle of PCR
Heat to >90 degrees to separated double stranded DNA
Use lower temperature to allow primers to hybridise to their target sequence
Allow DNA polymerase to extend the DNA
(Melting, reannealing, extension)
What is required for PCR
Short primers of 20 bases that hybridise to DNA flanking the region to be amplified. Template DNA dNTPs Buffer DNA polymerase
Where does the DNA polymerase come from in PCR
The DNA polymerase is Taq polymerase from Thermus aquaticus that grows in hot springs so can survive temperature changes
What is 3C
Chromosome Conformation Capture
DNA is digested in situ, crosslinked, and ligated together
Cross links are then reversed and ligated fragments are sequences
What is CRISPR/Cas9 based on
Immunity in archaea and some bacteria
What is CRISPR
Clustered Regularly Interspersed Short Palindromic Repeats
Short viral DNA sequences present in microbial genome that are transcribed into RNA and used as guides to direct the Cas9 nuclease to cleave the DNA of an invading virus
How is CRISPR/Cas9 used in experiments
Can do gene knockouts and allele replacements by preparing guide RNAs to target the desired genomic locus
What is better: mutated DNA or cell death?
Cell death: higher eukaryotes May induce apoptosis rather than live with potentially mutated DNA
Give 2 types of point mutation
Transition
Trans version
What is a transition mutation
When a purine is replaced by a different purine
What is transversion mutation
When a purine is replaced by a Pyrimidine (or vice versa)
What kind of damage can the DNA back bone undergo
It can be chronically modified or even broken
These breaks can be on 1 strand or both strands resulting in a double strand break (DSB)
What is the Ames test
Screening for mutagenic chemicals using salmonella
How does the Ames test work
A salmonella strain that cannot synthesis His is grown in the absence of His
Mutations that enable His synthesis result in Colony growth
Number of colonies is proportional to how mutagenic the chemical is
What is the safe threshold for a mutagenic agent
None
It is either mutagenic or not
Give 3 endogenous and exogenous causes of DNA lesions
Endogenous: ROS
Reactive chemicals
Chemical instability
Exogenous: UV light
Ionising radiation
Genotoxic chemicals
Give 4 possible errors that can occur during replication
Base mismatch
Deletion/ insertion
Low fidelity copying by trans lesion synthesis DNA polymerases
Incomplete replication
What gives DNA polymerase high fidelity
The incoming nucleotide must have properly paired with its Partner on the opposite strand before DNA polymerase adds it to the 3’ end of the previous base
If the polymerase incorporates the wrong nucleotide, how is the error fixed
Detected by polymerase itself
And corrected by 3’ to 5’ exonuclease activity associated with the enzyme, allowing the enzyme to back up. It removes the bad nucleotide and the. Polymerase drives the enzyme forwArds to redo
Discuss Huntington’s Disease
Autosomal dominant
Caused by insertion of a repeated sequence of CAG in Huntington gene
How many repeats are needed for symptoms to appear in Huntington’s
Less than 30 CAG repeats and they don’t have the disease
>40 repeats and they develop the cognitive and motor symptoms of the disease
How is anticipation exhibited in Huntington’s disease
Why might this be
Affected offspring often develop the disease at a younger age than affected parents as they have more copies of the repeats
One possibility: hairpin forms in repeat region chasing expansion here
Or
DNA repair systems may recognise misaligned bases in the hair pin and in the process of fixing it, expand the repeat sequence
What are the 3 major repair mechanisms for single stranded damage
Base excision
Nucleotide excision
Mismatch repair
How does each case of single stranded repair work overall
Recognition of incorrect/ damaged base
Removal/ repair of base or nucleotide
Filling gap with DNA polymerase and ligase
What are the 2 major classes of repair mechanism for double stranded break
Non homologous end joining
Homologous end joining
How can UV light affect DNA
Will the base have to be removed
Causes adjacent T residues to form diners through a cyclobutyl ring, deforming the double helix and obstructing DNA replication and RNA transcription
No it can be directly reversed
How can a mutated thymidine from UV radiation be corrected?
Photoreactivation of the T dimer, catalysed by DNA photolyase enzymes, converts the T dimer back to 2 single T residues
What happens if C deaminates spontaneously or by oxidising agents?
Forms U
U pairs with A so could result in a conversion if a CG base pair to a TA in later generations
When a C is mutated to a U, how does the body cope?
Uracil DNA glycosylase (UDG) recognises U in DNA.
It flips it out of the double helix and cleaves the glycosidic linkage to the deoxyribose
what is the result of UDG action
Creation of an “abasic site” (no bases are present)
What happens to an abasic site after the action of UDG
Recognised by a nuclease that cleaves the backbone producing a 3’OH upstream of the site.
This acts as a priming site for a DNA polymerase
What does the DNA polymerase do to the 3’OH during removal of an abasic site
DNA polymerase removes the abasic site and incorporates a C opposite the templated G
In
a) eukaryotes
b) prokaryotes
Which DNA polymerase is involved in removal of an abasic site
a) DNA polymerase β
b) DNA polymerase 1
Does UDG work for all mutated bases?
No it can only remove U but there are other DNA glycosylases that are specialised for other altered bases
Give an example of oxidative DNA damage
8-oxoGuanine
What happens if 8-oxoGuanine is left unrepaired
Where is the often found
What happens in the case of cytosine deamination?
Causes a GC to TA transversion
In human cancer cells
A specific glycosylase removes the oxidised base
Which method of DNA modification is not mutagenic
What is it used for?
Methylation on a C or A
To control gene expression
Discuss DNA methylation in bacteria
The Dam methyltransferase adds a methyl group to the A residue in the sequence GATC
This protects the DNA from digestion by the bacterium’s own restriction endonucleases
Discuss DNA methylation in eukaryotes
Only C (not A) can be methylated Occurs in CpG islands This switches off expression of nearby genes It is the basis of genomic imprinting
What is a CpG island
Regions containing repeats of CG
What is genomic imprinting
Eg?
When some genes show parent of origin
Eg: only the copy you inherit from your father is expressed, the maternal copy is methylated and silent
True or false:
Photo lyases repair the covalent linkage made between T’s (Pyrimidine dimers) when exposed to UV light in humans
False
These enzymes are not found in humans
What is NER
Nucleotide excision repair
The complex system for repairing Pyrimidine dimers and other base/nucleotide changes in DNA
How is the UV induced T dimer fixed in humans
Name 2 other enzyme types that are involved
The NER pathway detects the dimer and unwinds the DNA around the lesion.
The strand is cut up- and down-stream of the lesion, giving a short single stranded gap
The gap is filled by DNA polymerase and sealed by DNA ligase
Helicases and nucleases
What is xeroderma pigmentosum
An autosomal recessive syndrome
It is caused by the inherited mutation of genes that encode for enzymes in the NER
Give 3 symptoms of xeroderma pigmentosum
Extreme sensitivity to sunlight
Premature skin aging
Predisposition to skin cancers
What is the issue with a mutation causing an A to be swapped with a G
A new strand of DNA generated during replication is not methylated until some time after replication
Thus the newly generated DNA is hemimethylated with the parental template strand marked by methyl groups but not the new strand
How does mismatch repair machinery work
Preferentially repairs non methylated new DNA by excising a single strand of the new DNA that flanks the error (similar to the action of NER)
What are mutations in the mismatch repair system associated with in humans?
What is the name of the syndrome?
Inherited predisposition to cancer
Especially colorectal cancer
Hereditary nonpolyposis colorectal cancer syndrome
What are the 2 main mechanisms to ensure DSB repair
Non homologous end joining (NHEJ)
Homologous recombination
How does NHEJ work
Sticks broken ends of DNA back together which can restore original sequence but ends require some cleaning up.
May result in loss of DNA sequences at the joining site
What are the resting phases of the eukaryotic cell cycle
G1 and G0
When is NHEJ particularly important
In G1 and G0 (rearing phase)
What can loss of function mutations in NHEJ cause
Eg
Human Cancers
Eg mutations in DNA ligase IV are found in certain leukaemias
What does HR take advantage of
The fact that most organisms have more than a single copy of a given chromosome and the extra copy can act as a template for repair of a damaged chromosome
How does HR work in human DNA repair
DSB is processed to trim back the 5’ end, leaving an extended 3’ end which is recognised by a highly conserved protein.
This protein scans the genome for identical double stranded sequences and directs “strand invasion”, replacing one duplex strand with the single strand from the damaged DNA.
This allows prime replication from invaded end, copying the intact strand
Which proteins recognise the single stranded 3’ end in HR in
a) bacteria
b) eukaryotes
a) RecA
b) RAD51
What is a Holliday junction
Mobile 4 way junctions in DNA resulting from HR repair
What do Holliday junctions do
What allows this
Slide along DNA
The junctions are between homologous sequences
What enzyme facilitates branch migration of Holliday junctions in bacteria?
How does this work?
The RuvAB complex
It is a molecular motor that uses ATP to push the junction
How can a Holliday junction be resolved to 2 linear duplexes
In a way to restore parental configuration in both duplexes
Or
Crossed over configuration where DNA is swapped between the 2 original molecules
When would it be desirable to have a Holliday junction have a cross over configuration with the DNA swapped between the 2 molecules after HR?
During gene shuffling in meiosis to increase genetic diversity
Other than repair of DSBs, what else is HR useful for
Restarting stalled or broken DNA replication forks
What is the average of failed replication forks per cell cycle
10/ one eukaryotic cell cycle
Name 6 proteins involved in HR that will likely cause cancer if mutated
BRCA2 ATM Chk2 p53 Nbs1 Mre 11
Give an example of DSB processes that can have deleterious consequences
A translocation between parts of chromosomes 9 and 22
This is associated with Chronic Myelogenous Leukaemia
Give an example of the cell harnessing DSB repair for positive purposes
Elaborate
Antibody diversity generation
Number of different antibodies produced depends on number of combinations possible. Recombination events used to do this are error prone, thus increasing diversity
How is even more diversity added for antibody production
What mediates this process and when can its importance be seen
Terminal transferase adds extra nucleotides at the junction point, increasing diversity by being extra sloppy in how many bases they add
The NHEJ pathway
Mutations in NHEJ proteins result in immunodeficiency and failure to perform antibody diversity generation
How might targeting cellular mechanisms that maintain molecular stability be used in clinical medicine?
What is this technique called
Cancer cells often inactivate some of their own DNA repair mechanisms to acquire the mutator phenotype and become overly reliant on a smaller set of repair pathways. If these pathways are targeted, you can induce cancer cell death, with only minor harm to healthy cells
Synthetic lethality
Has synthetic lethality been effective?
Yes: Olaparib inhibits PARP.
PARP is highly effective in cancer patients with defective BRCA2 genes so PARP inhibition results in cancer cell death
Name a non hydrolysable analogue or lactose
IPTG
Name 2 small molecule inhibitors of RNA synthesis
Actinomycin D
Rifamycin
Describe the action of actinomysin D
Blocks all RNA synthesis immediately by binding tightly to dsDNA, intercalating between neighbouring GC base pairs
Does actinomysin D have any clinical relevance
Generally too toxic for clinical use but it’s inhibitor of the growth of rapidly dividing cells makes it an effective therapeutic agent against some cancers
Describe the action of rifamycin
Give its other name
An antibiotic that blocks all bacterial RNA synthesis by binding to β subunit of RNA polymerase
DOES NOT affect eukaryotic transcription, generally used to treat TB
AKA rifampicin
Name an RNA dependent DNA polymerase
Telomerase
What are restriction enzymes
Bacterial enzymes that cut DNA at/ near specific recognition sequences
Is DNA right or left handed?
Right handed!!!
Describe Z DNA
LEFT handed double helix (THE ONLY RIGHT HANDED ONE - Z is far from A and B)
Difference between A and B DNA
A is Shorter, more compact helical structure whose base pairs are not perpendicular to the helix axis
Which cancer can bevacizumab treat
Colon - blocks VEGF
What is CHK1 activated by
Double strand breaks and then will phosphorylate CDC25 (resulting in CDC25’s degradation)
CHK1 also phosphorylates WEE1, activating WEE1
What is the core complex of RNAi
RISC
RNA induced silencing complex
What is the protein domain present in Fos and Jun transcription factors
Leu zipper
What technique is used for measuring the on and off rate of binding between macro molecules
Surface plasmon resonance
What allosterically activates glycogen synthase
G-6-P
What enzyme is required to synthesise ketone bodies
Acetyl CoA acetyltransferase
