19.01.13 Mechanisms of mutations in DNA

Question 1

What is a mutation?

Answer 1

A permanent alteration in the DNA sequence

Question 2

How do mutations arise?

Answer 2

1) DNA damage 2) errors in DNA replication or recombination (see DSB repair section) 3) a failure to repair DNA damage

Question 3

DNA damage - what are the two classes based on origin?

Answer 3

Endogenous and exogenous

Question 4

1) DNA damage - what are the main causes?

Answer 4

1) Internal chemical events - depurination, deamination and oxidative damage (the majority of endogenous DNA damage arises from the chemically active DNA involved in hydrolytic and oxidative reactions). 2) Environmental agents - mutagenic chemicals (e.g. tobacco smoke) and certain types of radiation (UV and ionizing)

Question 5

Example of DNA lesion that needs repairing - missing base

Answer 5

• Depurination (removal of a purine base, adenine or guanine) - This is often caused by cleavage of the glyosidic bond btween deoxyribose and the base by acid and heat - If this isn’t repaired, then it generates mutations during replication

Question 6

Example of DNA lesion that needs repairing - altered base

Answer 6

• caused by ionising radiation or alkylating/oxidising/hydrolysing agents - Eg can get deamination of cytosine to uracil and if this isn’t corrected then you get the substitution of one base for another during replication

Question 7

Example of DNA lesion that needs repairing - Bulge due to deletion or insertion of a nucleotide

Answer 7

• Intercalating agents such as acridines, can cause addition or loss of a nucleotide during recombination or replication - insertion causes a DNA bulge - deletion causes an RNA bulge

Question 8

Example of DNA lesion that needs repairing - Linked pyrimidines

Answer 8

• UV radiation causes bonds to form between adjacent pyrimidine bases (can be C,T,U, but usually T) causing pyrimidine dimers - These distort the DNA structure, introducing bends or kinks which impede transcription and replication

Question 9

Example of DNA lesion that needs repairing - Single- or double-strand breaks

Answer 9

• Breakage of phosphodiester bonds by ionizing radiation or chemical agents, e.g. bleomycin

Question 10

Example of DNA lesion that needs repairing - Cross-linked strands

Answer 10

• Covalent linkage of two strands by bifunctional alkylating agents, e.g. mitomycin C - Interstrand DNA crosslinks (IDLs) makeup a particular subtype of DNA lesion because the IDL involves the covalent modification of both strands of DNA and these lesions can prevent DNA strand separation in DNA replication

Question 11

Example of DNA lesion that needs repairing - 3′-deoxyribose fragments

Answer 11

• Disruption of deoxyribose structure by free radicals leading to strand breaks

Question 12

2) Deficiencies in DNA replication

Answer 12

• Errors during replication are common - Major factor in determining spontaneous mutation rate - 3’→5’ exonuclease “proofreading” enzyme normally corrects mistakes but not all - estimated rate of 1x10-4 to 1x10-6 mutations per gamete for a given gene (i.e. 1x10-6 is 1 mutation per base in every million gametes)

Question 13

3) Defects in DNA repair

Answer 13

• DNA repair closely tied to cell cycle - Checkpoint mechanisms in place to ensure no errors before replication and division can occur - Failures in process cause mutations

Question 14

Defects in DNA repair - list 5 major pathways

Answer 14

1) Base excision repair (BER)
2) Nucleotide Excision Repair (NER)
3) MisMatch Repair (MMR)
4) Homologous Recombination Repair (HR)
5) Non-Homologous End Joining (NHEJ)

Question 15

Base excision repair (BER) - what is it and what damage does it repair?

Answer 15

• BER corrects DNA damage from oxidation, deamination and alkylation
• Principal repair pathway for the removal of oxidative damage
• DNA glycosylases recognise and remove the damaged bases by cleaving the N-glycosylic bond between the target base and the deoxyribose, releasing a free base and leaving an apurinic/apyrimidinic (AP) site

Question 16

Base excision repair (BER) - what are the main steps?

Answer 16

• It is initiated by a DNA glycosylase that recognizes and removes the damaged base, leaving an abasic site which is further processed by short-patch repair or long-patch repair
• Short-patch repair - involves single nucleotide insertion, and appears most common mechansim. AP endonuclease cleases phosphodiester bond immediately 5’ to AP site, generating 5’-sugar-phosphate and 3’OH ends. Get a single nucleotide gap which is filled by DNA polymerase and sealed by DNA ligase.
• Long-patch repair - involves a resynthesis patch of 2-13 nucleotides

Question 17

Nucleotide excision repair (NER) - what is it? and what are the 4 steps?

Answer 17

• Major repair system to UV damage (by removing pyrimidine dimers)
• Complex process involving more than 30 proteins to remove fragments of ~30 nucleotides
• 4 step process
  1) Detection of damage
  2) Nuclease excision of the section of DNA that includes and surrounds the error
  3) Filling of the resulting gap by DNA polymerase
  4) Sealing between old and newly synthesised DNA

Question 18

Nucleotide excision repair (NER) - what syndromes are assoicated with errors in NER process?

Answer 18

1) Xeroderma pigmentosum (XP)
2) Cockayne syndrome (CS)
3) photosensitive Trichothiodystrophy (TTD)
- These syndromes all have a basic defect resulting from mutation in a gene encoding one of several excision repair pathway proteins
- The “NER deficiency syndromes” share the common feature of extreme sensitivity to sunlight

Question 19

Mismatch repair (MMR) - what is it?

Answer 19

• process to detect and repair erros in DNA synthesis
• Recognises mismatched bases that are incorporated during replication
• These are corrected by excising a stretch of single stranded DNA that contains the error

Question 20

Mismatch repair (MMR) - what two heterodimers initiate process?

Answer 20

1) MutS-alpha - heterodimer of MSH2 and MSH6
- preferentially recognises mismatched base pairs [base-base] and small insertions/deletions from insertion deletion loops [IDL]
2) MutS-beta - heterodimer of MSH2 and MSH3
- binds mainly to larger looped out insertions/deletions [IDL >2bps]

Question 21

Mismatch repair (MMR) - what are the main steps of the process?

Answer 21

1) Mismatch recognised
2) MutSα or MutSβ recruits the MutL complex (heterodimer of MLH1 and PMS2)
3) Mismatched bases are exised
4) DNA polymerase fills the gap
5) DNA ligase seals the strands

Question 22

Mismatch repair (MMR) - what happens when this process is defective?

Answer 22

• Decrease in apoptosis and increase in cell survival
• Increase in damage-induced mutagenesis
• This provides a growth advantage to cell and you get tissue-specific cancers
• Often lead to replication errors such as replication slippage - commonly seen in microsatellite instability

Question 23

Microsatellite instability - what is it?

Answer 23

• Microsatellites are short DNA motifs (1-6 bases repeated)
• Present in coding and non-coding regions of the genome
• Implicated in most cancers
• MI causes alterations in the length of tandem nucleotide repeats, which in turn cause temporary self’complementary insertion deletion loops during replication
• If these are not corrected by MMR then they can generate missense or frameshift mutations in coding genes
• Causes dysfunction proteins
• MMR defect can cause 100 to 1000 fold increase in mutation rate

Question 24

Give example of inherited syndrome with MMR defects

Answer 24

1) Lynch syndrome (HNPCC)
2) Lynch syndrome variants (brain tumours in some colon cancer patients)
3) Muir Torres syndrome (rare skin tumours)
4) Sporadic MLH1-deficient colon cancers

Question 25

Double strand break (DSB) repair - name two processes used to repair this type of damage

Answer 25

1) Homologous recombination repair (HRR) 2) Non-homologous end joining (NHEJ)

Question 26

Homologous recombination repair (HRR) - what is it?

Answer 26

- Repairs DSBs - Utilises sister chromatids during G2 phase of cell cycle - Single strand from homologous chromosome invases damaged DNA and acts as a template for accurate repair - Requires sequence identidy of over 300bp - Normally occurs during meiosis - Involves crossing over and exchange of allelic or nonallelic DNA fragments often involving repeated sequences (for example, non-allelic homologous recombination, gene conversion) - Normally occurs through Double Holliday Junction (HJ), but less commonly with synthesis dependent strand annealing (SDSA)

Question 27

NAHR - what is it?

Answer 27

- Most common type of disease associated genome rearrangement - Get unequal crossover due to recombination between non-allelic homologous regions (LCRs) - Leads to deletion, duplication or translocations - Can be intra-inter-chromatid or interchromosomal

Question 28

Gene conversion - what is it?

Answer 28

- Describes a *nonreciprocal* transfer of sequence information between a pair of allelic or non-allelic DNA sequences - The donor sequence remains unchanged - Only the acceptor sequence is modified - Gene conversion tracts are usually short, 200bp–1kb - Examples in disease include: 1) SMA (*SMN1* gene and pseudogene *SMN2* in inverted repeat region on chromosome 5q13.2 from conversion of exon7 and intron 7 respectively resulting in exon skipping) 2) VWD (between *VWF* gene on chromosome 12p13.3 and its pseudogene on chromosome 22q11.22 - In nearly all cases of disease causing interlocus gene conversions, the acceptor and donor pair are on the SAME chromosome, however VWF is a rare exception

Question 29

What other process is homlogous recombination involved in?

Answer 29

- Also repairs collapsed or broken replication forks - Process is called break-induced replication (BIR) - If the broken end invades a homologue instead of a sister molecule or the repair involves homologous sequence in a different chromosomal position it can lead to LOH, translocation, duplication or deletion, thus constituting an alternative mechanism for NAHR

Question 30

Non-homologous repair (NHR) - what two subdivisions are there?

Answer 30

- Replicative and non-replicative mechanisms

Question 31

Non-replicative mechanism for NHR - Example 1 -NHEJ

Answer 31

- Non-homologous end joining (NHEJ) - Get ligation of any two DSBs (homology not required) - Regions can be from different genes or chromosomes - Complex of two proteins, Ku and DNA-dependent protein kinase, bind to the ends of a double-strand break - After formation of a synapse in which the broken ends overlap, Ku unwinds the ends, by chance revealing short homologous sequences in the two DNAs, which base-pair to yield a region of microhomology - The unpaired single-stranded 5′ ends are removed, and the two double-stranded molecules ligated together - As a result, the DSB is repaired, but several base pairs at the site of the break are removed

Question 32

Non-replicative mechanism for NHR - Example 2 -Breakage-fusion-bridge cycle

Answer 32

- major role in amplification of cancer - Upon replication of a chromosome that has lost its telomere due to a DSB, there will be two sister chromatids that lack telomeres - The chromatids will fuse creating a dicentric chromosome - During anaphase, the two centromeres will be pulled to separate nuclei, causing eventual breakage of the dicentric chromosome - The break will lead, after replication, to new ends that lack telomeres, so that these ends will again fuse, forming a new dicentric chromosome and a cycle is established - Random breakage causes large inverted duplications, and repeated cycles could lead to amplification of the inverted repeat - The cycle will cease when the chromosome acquires a telomere.

Question 33

Replicative mechanism for NHR - Example 1 - replication slippage

Answer 33

- Form of mutation that leads to either a trinucleotideor dinucleotide expansion or contraction during replication - A slippage event normally occurs when a sequence of repetitive nucleotides are found at the site of replication (i.e. tandem repeats) - During replication, a length of lagging-strand template becomes exposed as a single strand - The 3' primer end can move to another sequence showing a short length of homology on the exposed template; this move might occur owing to the formation of secondary structures in the lagging-strand template - Lagging strand synthesis can continue after having failed to copy part of the template - Many human diseases have been reported to be associated with trinucleotide repeat expansions including HDs

Question 34

Replicative mechanism for NHR - Example 2 - fork stalling and template switching (FoSTeS)

Answer 34

- Template switching could occur between different replication forks - An exposed single-stranded lagging strand template can acquire a secondary structure - This can block the progress of the replication fork, forcing the 3' end then align on another exposed single-stranded template sequence on another replication fork that shares microhomology - Thus causing duplication, deletion, inversion or translocation, depending on the relative position of the other replication fork - FoSTeS is now superseded by MMBIR.

Question 35

Replicative mechanism for NHR - Example 3 - microhomology-mediated break induced replication (MMBIR)

Answer 35

- MMBIR is similar to BIR in HR but requires little homology - MMBIR postulates that the 3′ end from the collapsed fork will anneal to any single-stranded template with which it shares microhomology and that occurs in physical proximity to the 3′ DNA end, and initiate DNA synthesis and a low-processivity replication fork - This annealing reaction requires very little homology, so that annealing will occur with the sister molecule either in front of or behind the position of the fork collapse, leading to deletion or duplication respectively, and in either orientation, giving the opportunity to form an inversion - Microhomology might also be found in a different chromosome, leading to translocation