1
Q
What is a mutation?
A
Change in nucleotide sequence of DNA that may alter the function of a protien
2
Q
What is the effect of a mutation?
A
Mutation can occur anywhere in the DNA. If it occurs WITHIN a gene, it may or may not have an impact on the encoded gene product or its expression
3
Q
What do mutations outside of ‘coding’ regions affect ?
A
- Binding sites
- Alternative splicing (way exons are brought together)
4
Q
What allows for variation?
A
The introns
5
Q
What do alleles arise from?
A
Mutations
6
Q
What effects can mutations in genes have?
A
- Prevents protein from forming e.g lack of dystrophin (muscular dystrophy)
- Reduces quantity e.g. slow blood clotting in heamohpila A
- Reduces protein efficiency e.g. sickle cell anaemia
- changes protein function e.g. extra aa’s alter function in Huntingtons disease
7
Q
Are inherited mutations present in multifactorial disorders?
A
- They might be, they also might not..
- They are responsible for single gene disorders though like CF
8
Q
Are non inherited mutations present in multifactorial disorders?
A
YES! They CONTRIBUTE to multifactorial disorders such as cancer, heart disease, diabetes, Alzheimers, predisposition diseases
- Are also cumulative (mutations cumulate with age)
- Also UV
9
Q
What are the major classes of mutation?
A
- Point Mutations
- Large scale mutations
- Trinucleotide repeat expansion
10
Q
What are the types of point mutations?
A
- Base substitutions ( missense, nonsense, silent)
- Single base deletion/insertion (frameshift mutations)
11
Q
What is a missense (point) mutation?
A
Where there is a single amino acid change
12
Q
What is a nonsense (point)mutation?
A
Mutation change in a base that codes for a STOP codon early- results in non functional protein
13
Q
What are the different types of large scale mutations?
A
- deletion/insertion (silent)
- Duplication-swapping DNA sequence around ( loss of function)
- Duplication ( gain of function)
- Translocation ( conditional- if you get a temperature and loss of protein from that)
14
Q
What is an example of a base substitution (real example) ?
A
Deamination (C–>U)
- results in one normal strand and one mutated strand
15
Q
What is the process (example) where there can be base substitution/ deletion leading to a frameshift mutation?
A
Depurination ( either loss of A or G)
- Strand deletes nucleotide because no base anymore and it fails to seal
- Hence frameshift mutation
- can result in short protein
16
Q
Where is the mutation in the dystrophin gene?
A
79 exon
17
Q
What are DMD and BMD commonly caused by?
A
- Mutations resulting in large deletions
18
Q
How come BMD and DMD produce different symptoms?
A
- DMD allows NO FUNCTIONAL protein to be made from mutation but BMD still allows some function…but reduced
19
Q
What does a BMD mutation not change?
A
The frame of the gene
20
Q
What type of mutation occurs with a truncated protein (non functional) ?
A
Nonsense mutation (STOP codon)
21
Q
What type of mutation occurs with a protein that has latered function due to an amino acid change?
A
Missense mutation
22
Q
When do inversions occur (large scale chromosomal mutations)?
A
When a broken segment is inserted in reverse order
23
Q
When does reciprocal translocation occur (large scale) ?
A
When NON HOMOLOGOUS chromosomes exchange segments
24
Q
When do duplication and deletion occur? (large scale)
A
When HOMOLOGOUS chromosomes break at a different point and swap segments
25
Which method can detect large scale mutations in evolution?
FISH
| - Fluorescent In Situ Hybridisation (in chromosomes)
26
What are some things that can cause mutations?
- Retroviruses and transposons (loss of function and duplication)
- Spontaneous (occurs naturally)
- Induced (require a mutagen)
27
What effect does Retroviruses and transposons have in causing mutations?
- They may cause disease from mutations
| - Have a role in evolution
28
What effect does spontaneous mutations have ?
- Arises in cells at low frequency
- Errors in DNA replication (due to tautomers)
- Spontaneous lesions or damage (e.g. depurination, deamination of C)
29
What are examples of induced mutation substances?
- Chemical (e.g. base analogues, intercalating agents, base modifiers (Aflatoxins, Benzopyrenes, nitrites) )
30
What is an example of spontaneous mutation?
If thymine is incorperated into DNA during replication while it is in its rare tautomeric form, it will pair with G rather than A (so base substitution)
31
What is an example of induced mutation?
Melanoma from UV
| - Pyrimidine dimer (T-T) forms--> cyclobutane ring
32
What occurs when DNA replacing enzymes can't function?
- Mostly cancers
| e. .g BRCA1--> homologous repair process fails
33
What are the different types of spontaneous alterations to bases that require DNA repair?
- Oxidative damage
- Hydrolytic attack
- Uncontrolled Methylation
34
What is oxidative damage?
- It is metabolic and Guanine more susceptible
35
What is a hydrolytic attack?
Where it CLEAVES the chemical bonds in DNA
36
What is uncontrolled methylation?
ALKYLATION of bases (attachments of methyl groups)
37
What is depurination?
Spontaneous loss of purine bases (A, G) by hydrolysis (N-glycosyl linkages to deoxyribose hydrolyse )
- Sugar backbone bond broken by hydrolytic attack (H2O) --> Guanine detaches
- No base on the backbone now
38
What is deamination?
- The spontaneous loss of amine group on the CYTOSINE
- this loss changes molecule to Uracil
- this leads to problems with the DNA structure AND when DNA replicates
39
What is another way bases can be damaged?
- UV radiation -->
| - Produces covalent linkage b/w two adjacent pyrimidine bases in DNA to form Thymine dimers
40
What are thymine dimers?
- Covalent linkages on C-C bonds form lesions and later DNA structure
- (note a similar dimer can form between any two pyrimidine bases--> C or T in DNA )
41
What can thymine dimers cause?
- Sunburn (Damaged DNA)
- melain production
- Melanoma
42
What can base alteration changes lead to if uncorrected when replicated?
- Can lead to deletion (of one or more base pairs) OR can lead to base pair substitution in daughter DNA chain
43
What occurs in deamination (mutation)?
- There is loss of nucleotide pair
- When replication machinery encounters missing purine on template, it SKIPS to the NEXT NUCLEOTIDE
- This leads to deletion
44
What happens when one strand of DNA is damaged?
- The repair machinery looks at the non damaged strand to copy the info and repair
45
What is it about DNA that allows for such good repair mechanisms?
The double stranded nature of it
46
What are the two common DNA damage repair pathways?
- BASE EXCISION REPAIR
| - NUCELOTIDE EXCISION REPAIR
47
What are the three general steps that occur in damage repair pathways?
1. Damage excised
2. Original sequence restored by using un-damaged strand (by DNA polymerase) as the template
3. Remaining break sealed by DNA ligase
48
How do the two pathways (base excision and nucleotide excision) difer?
- Differ in the WAY that they remove damage from the DNA
49
What is base excision repair step 1?
1. Group of enzymes (GLYCOSLYASES) recognise specific type of altered base in the DNA
- Then catalyse its HYDROLYTIC removal
- To detect the base in helix, altered nucleotide is 'flipped out' by glycosylases
50
How is a base detected in base excision repair?
The flipping out method,which hallows it to look at all angles for damage and evaluate the status of each base
51
In base excision repair, what happens when an enzyme finds the damaged base that it recognises?
- Glycosylases remove the base from the sugar
| - This results in a 'missing tooth'
52
What can the 6 types of glycosylases remove?
- Deaminated C's
- Deaminated As
- C_-C-->C-C (mistake in bond)
- bases with open rings
- alkylated or oxidised bases
53
In step 2 of base excision repair, what recognises the missing tooth created by glycosylases?
AP endonucleases
54
What does an AP endonuclease do?
- Cuts the phosphodiester backbone
55
What happens in step 3 of base excision repair?
After the phosphodiester backbone is cut, DNA polymerase adds new nucleotides THEN ligase seals the nick
56
How are depurinations repaired?
- Deoxyribose sugar has no base so...
| - They come in at step 2 of base excision repair where they are DIRECTLY repaired by AP ENDONUCLEASES and ligased
57
What does base excision repair mainly work on?
Deamination process
58
What is nucleotide excision repair?
- for LARGER regions of damage in the DNA double helix
- can include bulky lesions created b y carcinogens (diesel exhaust, tobacco smok)
- also can include pyrimidine dimers (T-T, T-C, C-C) caused by UV
59
What occurs in step 1 of nuecleotide excision repair?
MULTIENZYME COMPLEX scans DNA for DISTORTION in DNA helix (instead of looking at specific change in base)
60
What happens once it finds the lesion site? (step 2)
- Cleaves the ph.diester backbone of ABNORMAL STRAND on BOTH SIDES of the distortion
61
What happens in step 3 of nucelotide excision repair?
- DNA helcase peels away single stranded ologonucleotide containing lesion
62
What occurs in steps 4 and 5 of nucleotide excision repair respectively (What is the large gap in the helix repaired by?)
- DNA polymerase and DNA ligase repair the gap
63
What is an alternative to base excision repair AND nucleotide excision repair processes?
- DIRECT CHEMICAL REVERSAL REPAIR
64
What is direct reversal repair used for?
- Certain highly mutagenic or cytotoxic lesions e..g Alkylation lesion O6 (methylgnuanine has the methyl group removed by direct tansfer to cysteine residue IN repair protein.... repair protein dies in the reaction
65
What does the Methyltransferase (MTase) protein do?
- Accepts the CH3 on cystine residues from alkylated guanine nucleotide (restores normal guanine, MTase INACTIVATED, no DNA cleavage or ligation required)
66
What occurs in xerodoma pigmentation?
- Defect of nucleotide excision repair
| - Nucleotide excision repair enzymes are mutated
67
Why is transcription coupled DNA repair important?
- Because it ENSURES cell's MOST IMPORTANT DNA is repaired first ( most urgently needed to be repaired)
68
What does transcription coupled DNA repair do?
- Links RNA polymerase to nucelotide excision repair pathway
- RNA polymerase (transcribes DNA--> RNA) STALLs at DNA lesions and DIRECTS repair machinery to to those sites
- Reaction in eukaryotes is MUCH more complex than proaryotes because genes longer in eukaryotes
- Targets repair to genes that are ACTIVELY trasnscribed into mRNA
69
What happens when transcription coupled DNA repair is is defective?
- If there is ANY mutation in ANY gene, RNA polymerase will STOP (permanently stall) and not continue instead of repairing
- As a result DNA sensitivity to UV occurs as there is no repair systems
- Example of this is Cockayne syndrome (retardation, death by 20)
70
What does a cell do if DNA suffers EXTREME damage?
- Back up DNA polymerases come in called TRANSLESION POLYMERASES to replicate through DNA damage
71
What do the translesion polymerases lack?
- Lack exonucelolytic proofreading activity
| - Only adds one or a few nucelotides before it falls off and replicative bit follows from there
72
How come translaesion pomyerases as back up is risky in extreme cell damage?
- Because they are repsonsible for the most base substitution and single nucleotide deletion mutations (that accumulate in genomes)
- obviously can produce more mutations on damaged DNA but can ALSO produce mutations on undamaged DNA
73
How are breaks repaired (2 methods) ?
- Homogolous recombination
| - Non homologous end joining
74
What occurs in non-homologous end joining?
- Broken ends brought together and rejoined by DNA ligation (common in mammalian somatic cells)
- there will be lots of genomic scars where DNA has been roughly glued back together by this process (but it is okay because we have lots of introns)
75
What is non homologous end joining in more detail? (Ku etc)
- Ku protein (heterodimer) grasps broken chomosome ends
- Additional proteins hold broken ends together while they are processed
- Then ends are joined covalentely
76
What is the issue with non homologous end joining?
- There is no way to tell if broken ends being covalentely joined were ORIGINALLY next to each other or not
- this can result in chromosomes with 2 centromeres or chromosomes lacking centromeres completely
77
What does the specialised structure of telomeres prevent?
Prevents the natural ends of chromosomes from being mistaken for broken DNA and "repaired" in this way
78
What can eukaryotic cells do to maximise effectiveness of DNA repair enzymes?
- DELAY the progression of the cell cycle until DNA repair is complete
- can block entry from G1-->S phase
- can slow down the S phase
- can block transition from G2-->M
There is also increased synthesis of DNA repair enzymes
79
When does the cell cycle STOP?
- When damaged DNA is detected
80
What is the ATM protein?
- Kinase needed to generate intracellular signals that sound the alarm in response to SPONTANEOUS DNA damage
(links signals between DNA damage machinery and the cell cycle)
81
What is homologous recombination?
Exchange of DNA strands between pair of homologous duplex DNA sequences (segments of double helix that are very similar or identical in nucleotide sequence)
- one strand acts as a template to restore other strand
82
What does homologous recombination correct?
- DNA replication forks that become stalled during replication or even if they just get broken
83
What does hoomologous recombination take part in?
- Accurate repair of double stranded breaks in DNA
- Genetic exchange in meiosis
- Mechanic role to make sure chromosomes segregate accurately during meiosis in fungi, plants and animals
84
How do double stranded breaks occur?
Environmental damage
| DNA replication were fork becomes stalled-occurs in nearly every round of DNA replication
85
Is non homologous joining or homologous recombination better for DNA?
- Homologous repair is BETTER!
- Results in virtually no mutations
- Accurate
- Requires daughter duplex to be nearby to allow for swapping over of information for repair
86
What are the first 3 steps in homologous recombination?
1. Exonuclease chews/retracts the 5' ends to leave 3' overhang (ss DNA now) and to get strands mixing with each other
2. The 3' overhang invades the other sister chromatid if it is separated, so base pairs with other sister chromatid
3. EXTENSION occurs of INVADING STRAND and DNA synthesis occurring. Polymerase gets on the chromatid, extends it, adds bases on (branch migration) then it falls off/apart
87
What are the last 3 steps in homologous recombination?
4. New DNA (green) can meet up with the old strand that was previously chewed up
5. There is a gap left but DNA polymerase fills it
6. Ligase then ligates the strands and repair is done
88
What does the replication fork do when it reaches a nick?
- Stops
- It adds bases onto one strand fine but the other strand will fall apart (disconnected and unextended- so one broken and one intact daughter chromosome)
- SO the broken one uses the intact one to fix it--> homologous recombination
89
When does homologous recombination take place?
AFTER DNA replication but before the cell has divided
90
What is the process of strand invasion?
RecA protein (EColi) intertwines the ssDNA and DNA duplex in SEQUENCE DEPENDENT manner (no particular place )
- DNA single strand searches duplex for homologous sequences in triplets
- Single strand DISPLACES one strand of the duplex and forms base pairs with other strand resulting in HETERODUPLEX
91
What is a heteroduplex?
- Pairing of two different strands from two different DNA molecules
92
At least how many nucleotides are needed for a strand invasion to occur?
Needs at least 15 nucleotides for a strand invasion to occur
93
What is branch migration in homologous recombination?
- Occurs after strand invasion
- Point of strand exchange can MOVE after strand invasion so this is termed branch migration
- Specialised proteins make sure that the branch is going in one direction
- New double helix (branch) starts migrating and unzippering the rest of the double helix in one direction
94
How does the strand invasion REACTION occur and what does the filament cataylse?
- RecA uses ATP to bind tightly to the ssDNA forming a DNA protien filament (BOTH the single strand AND double stranded DNA-so 3 stranded complex)
- Filament catalyses reaction that takes off the complementary double helix, starts separating it, and allowing the extra strand to invade
95
What is the outcome of Meiosis?
4 genetically unique haploid cells
96
What is the outcome of mitosis?
2 genetically identical diploid cells
97
What are homologous chromosomes?
One from mother, one from father, similar in length, gene positions, and centromere location
98
What are sister chromatids?
Two IDENTICAL copies of a SINGLE chromosome that are connected by a centromere
99
When does crossing over occur in meiosis?
In prophase I (b/w prophase I and metaphase I)
100
How does the DNA repair systems account for distinguishing paternal and maternal apart?
- Randonly selects one and changes the ds DNA to that one
- Called gene conversion (from one allele to another)
- Only limited to small sections of DNA (doesn't conform to Mendelean genetics)
101
What is recombination?
- The natural formation in offspring of genetic combinations not present in parents by the processes of crossing over of homologous sequences
- repair like mechanism
- diverse genetic population
- extremely similar to homologous recombination repair
102
How is crossing over mechanism different to the repair mecanism?
- In crossing over, our proteins MAKE the double stranded break on purpose
- Spo11 and Mre11 nuclease complex jump onto chromosome and cut it (just like DNA break)
- Then the rest of the steps are similar to homologous recombination
103
Can too much or too little homologous recombination cause cancer?
YES!
104
What occurs in 90% of cases of meiosis?
-After extension and rebinding to chromosome, binding to original chromosome occurs and restoration occurs with NO CROSSOVER
105
What occurs in 10% of cases of meiosis?
- Looped out part binds onto the rest of the chromosome
- So you end up with extension BOTH ways
- This produces a holiday junction
- Junction migrates and is then resolved by cutting the DNA strands
106
What determines whether you get crossing over or not?
Where the cleavage takes place
107
What are the 4 steps in determining crossed or uncrossed?
1. homologous chromosomes nicked
2. Strand invasion
3. Branch migration and heteroduplex formation
4. Endonuclease cleavage site important to separate chromosomes
(a) Crossed strand cleavage= non-recombinant chromosomes
(b) Rotation (isomerisation), cleavage of uncrossed strands=recombinant chromosomes
108
What type of chromosomes result from crossed strand cleavage?
Non recombinant chromosomes (no crossing over )
109
Which type of chromosomes results from rotation (isomerisation) ?
- Cleavage of uncrossed strands = recombinant chromosomes (crossing over occurs)
