L7: DNA Mutability and Repair Flashcards
what are the 2 categories of mutations
- detrimental
- beneficial
2 categories of mutations - detrimental
- more common
- most are deleterious
- cell cycle gene defects can lead to cancer
2 categories of mutations - beneficial
genetic variation required for evolution
what are the 2 consequences of mutations?
- regulatory or coding sequences of genes are altered and gets passed to progeny
- chromosomal structural changes that can then impede DNA replication and affect cell survival
what are the 3 sources of mutations?
- DNA replication errors
- Chemical/environmental damage to DNA
- insertions generated by DNA elements (Transposons)
DNA replication errors - cause
- nitrogenous base pairs are usually found in one tautomeric form
- instances of rare tautomer formation changes results in mispairing of bases
DNA replication errors - tautomeric form of nitrogenous bases
- enol form: has a hydroxyl group
- keto: double bond on O
- usually C pairs with keto-G
- but if it changes to enol-G, it will fit in with T and DNA Pol cannot detect the mispair
DNA replication errors - what are the classes of mutations
- transition
- transversion
- point mutations
- frameshift mutations
- chromosomal rearrangements
DNA replication errors: classes - transitions
- same group different identity
- purine converted to purine (A ⇋ G)
- pyrimidine converted to pyrimidine (T ⇋ C)
DNA replication errors: classes - transversion
- group is completely different
- purine converted to pyrimidine (A → T/C or G → C/T)
- pryrimadine converted to purine (T → G/A or C → G/A)
DNA replication errors: classes - point mutation
- single base change
- has 3 types:
1. missense mutation
2. silent mutation
3. nonsense mutation
DNA replication errors: point mutation - missense mutation
- changes the amino acid sequence of a protein
- damage depends on the type of amino acid being produced
DNA replication errors: point mutation - silent mutation
- does not change the amino acid sequence of a protein
- least detrimental since it protein function does not change
DNA replication errors: point mutation - nonsense mutation
- amino acid-specifying codon is changed to a stop
- most detrimental since its most likely to create a non-functional protein
DNA replication errors: classes - frameshift
- mutation that alters the meaning of all downstream codons
- detrimental
DNA replication errors: classes - chromosomal rearrangements
- larger in scale
- can result in additions or deletions of chromosomal regions
DNA replication errors - chromosome-level mutations
- inversions
- translocation
- deletion
- duplication
DNA replication errors: chromosome-level mutations - inversion
sections of a chromosome break and rotate before rejoining
DNA replication errors: chromosome-level mutations - translocation
chromosome piece breaks and attaches to a different chromosome
DNA replication errors: chromosome-level mutations - deletion
- segment of a chromosome is lost
- detrimental since it causes the loss in thousands of genes
DNA replication errors: chromosome-level mutations - duplication
- additional copies of a chromosome segment is gained
- damage depends on molecular structure of the gene being effected
- genes that require a specific amount to be effective will make the mutation detrimental
chromosome-level mutations: duplication - example of how it can be detrimental
- Charcot-Marie-Tooth (CMT) Disease
- caused by partial duplication of chr 17 including the PMP 22 gene (encodes myelin protein)
DNA replication errors - DNA microsatellites
- sequences in DNA usually don’t code for genes (not always)
- repetition of 3 or 4 nucleotides over and over again
- hotspots for mutations and can cause diseases
DNA replication errors: DNA microsatellites - why is it a hotspot for mutations?
- it can cause slippage of replication machinery
- some new DNA will slip and ‘bow out’
- replication will continue but it creates an extra repeat in the synthesized strand
- but can happen to the template, resulting in a loss of genetic material
DNA replication errors: DNA microsatellites - what’s an example of a disease that can be caused by this?
- Huntington’s Disease: degradation of nerve cells in the brain
- if errors create an increase in CAG repeats, individual will get an earlier onset of the disease
mismatch repair mechanism
- corrects mismatches incorporated due to DNA replication
- if mutation escapes proof-reading enzyme, fast-acting repair mechanisms must be in place to correct the error
mismatch repair mechanism - why must repair happen fast?
- during the first round of replication, if there is an error, a ‘bump’ is then created and detected (A-T vs A-G)
- if it escapes the proof-reading enzyme, the second round of replication will have the correct base pairing (A-T vs G-C)
- results in mutation no longer having bump and cannot be detected
mismatch repair mechanisms - E. coli
- uses MutS
- will then recruit MutL and MutH
mismatch repair mechanisms: E. coli - what is MutS?
- a mismatch repair protein that acts as a dimer
- it scans DNA and recognizes distortion due to mismatch
- binds to ATP and creates a kink in the duplex
- will then recruit MutL (endonuclease activity) and MutH (exonuclease activity - needs to terminal end to work)
mismatch repair mechanisms: E. coli - what is hapspens after MutS recruits MutL and MutH?
- they bind to the DNA and create a nick one one strand
- exonuclease (MutH) degredes DNA beyond mismatch (broad cut)
- DNA Pol III and DNA ligase fills in the space
mismatch repair mechanisms: E. coli - how does the cell know which strand to repair?
- enzyme Dam methylase methylates DNA
- it methylates A on both strands
- but immediately following replication, the DNA is hemi-methylated with only the parental strand haying methyl marks
- MutH only nicks the non-methylated strand (newly synthesized)
chemical/environmental damage to DNA - what are the types?
- hydrolysis
- radiation
chemical/environmental damage to DNA - hydrolysis
- interactions with water
- can cause deamination
chemical/environmental damage to DNA: hydrolysis - deamination of cystine
- water reacts with cystine and results in the loss of a NH2 group and creates uracil
- this makes the nucleotide convert to RNA form
- creates a G-A mutation (transition)
chemical/environmental damage to DNA: deamination of cystine - would it be worse if it was a thymine changing instead?
- no, consequences wouldn’t be that bad
- bc if T changes to U, A will still fit inside it (T-A is the DNA equivalent to U-A in RNA)
chemical/environmental damage to DNA: hydrolysis - what does this answer?
- the question: why does DNA have T but RNA have U?
- possible answer: C → U mutation in DNA will make the cell know something is wrong since U is part of RNA
- if U was in DNA naturally, the cell wouldn’t be able to see there is a mutation
chemical/environmental damage to DNA - radiation
- UV light
- γ-radiation and X-rays
chemical/environmental damage to DNA: radiation - UV light
- absorbed strongly by bases
- can result in chemical fusion of neighboring pyrimidines (“dimers”) - also called thymine/pyrimidine dimers
- incapable of base-pairing and stall DNA synthesis
chemical/environmental damage to DNA: UV light - what are dimers?
- two pyrimidine structures near each other form covalent linkages between each other
- forms a cyclobutane ring and cannot interact with their partnered bases on the other strand
chemical/environmental damage to DNA: radiation - γ-radiation and X-rays
- cause double-stranded DNA breaks
- can be lethal to cell since intact chromosomes are required for DNA replication
chemical/environmental damage to DNA - how to repair broken DNA?
- direct reversal
- excision repair
- recombinal repair
- translesion DNA synthesis
chemical/environmental damage to DNA - direct reversal
- for Pyrimidine dimers (UV light)
- uses photoreactivation
chemical/environmental damage to DNA: direct reversal - photoreactivation
- enzyme DNA photolyase uses light energy to directly repair pyrimidine dimers
- does it by break the abnormal covalent bonds between the pyrimidines
chemical/environmental damage to DNA - excision repair
- two types:
1. base excision repair: replacement of specific, single base
2. nucleotide excision repair: stretch of nucleotides are replaced in DNA (more broad)
chemical/environmental damage to DNA: excision repair - base excision repair
- for small base modifications (deamination - hydrolytic damage)
1. enzyme Glycosylase hydrolyzes glycosidic bond of damaged base
2. AP (“apurinic” or “apyramidic”) endonuclease removes abasic sugar
3. repair DNA Pol and DNA ligase restores correct nucleotide
chemical/environmental damage to DNA: excision repair - nucleotide excision repair
- for large DNA distortions (UV light)
- single stranded DNA segment containing lesion that distorts duplex (either side of DNA damage)
- enzymes in E. coli: UvrA-UvrB complex, UvrA, UvrB UvrC, UvrD
- higher eukaryotes (humans) have these as well, but different names
chemical/environmental damage to DNA: nucleotide excision repair - E. coli
- UvrA-UvrB complex detects duplex distortion
- UvrA subunits are released
- UvrB dimer melts duplex
- UvrC nicks either side of lesion
- UvrD helicase unwinds and removes strad
chemical/environmental damage to DNA: nucleotide excision repair - repair enzyme mutation
- results in Xeroderma pigmentosum
- recessive disease caused by inability to repair UV-damaged DNA
- results in skin lesions and higher rates of skin cancer
chemical/environmental damage to DNA - recombinational repair
- used when both strands are damaged (radiation)
- done via non-homologous end joining (NHEJ)
chemical/environmental damage to DNA: non-homologous end joining (NHEJ) - how does it work?
- late resort and error-prone (mutagenic)
- DNA ends are processed for ligation (chewed off) by a variety of factors
- processing adds nucleotides and can introduce insertions or deletions
chemical/environmental damage to DNA - translesion DNA synthesis
- for UV damage
- last resort since its error-prone and mutagenic
- if lesion is not repaired and stalls DNA Pol, translesion synthesis can bypass the error altogether
chemical/environmental damage to DNA: translesion DNA synthesis - how does it bypass the error
- some parts of DNA are not synthesized (bc of dimer) division results in DNA break so chromosome is not replicated
- but one cell has too much DNA and another has too little
- the cell then employs a special class of translesion polymerases
chemical/environmental damage to DNA: translesion DNA synthesis - special class of translesion polymerases
- DNA Pol IV or V
- can add nucleotides independently of base-pairing
- not involved in everyday replication since they do not put in the correct nucleotide
- and DNA Pol III is not used since it cannot deal with this break
chemical/environmental damage to DNA: translesion DNA synthesis - how does the translesion polymerase access the DNA?
- the sliding clamp of DNA Pol III is ubiquinated
- this serves as a signal to recruit translesion polymerase which adds nonspecific nucleotides
- replicative DNA Pol can then resume synthesis
chemical/environmental damage to DNA: non-homologous end joining (NHEJ) and translesion DNA synthesis - why does are they used if they are error-prone
its better than not repairing the break since this can block replication or chromosomal loss resulting in death or tumor formation
