BMS1062 - W10: Recombination, Repair and Mutations

What is a conservative mutation?

Mutations which result in the same kind of amino acid

• e.g. original amino acid = Glu. Mutated amino acid = Asp. Both are Acidic Amino acids.

What is a non conservative mutation?

Mutation where new amino acid is of a different type to the original amino acid

What is a mutation?

Any change in the nucleotide sequence of DNA

What are germline mutations?

Inherited mutations

Mutations inherited form parents

• present in all nucleated cells of the body (including germ cells)

• responsible for single gene disorders (e.g. cystic fibrosis)

• may or may not be present in multifactorial disorders (cancer, heart disease, diabetes, etc.)

What are somatic mutations?

Non-inherited mutations

• affect only the mutant somatic cell and its descendants 

• will not be transmitted to offspring 

• contribute to multifactorial disorders (e.g. cancer, heart disease, alzheimer’s)

Point mutations:

Occur usually in a single base (base substitution or base deletion/ insertion)

• missense mutation - change in an amino acid

• Nonsense mutation - causes a stop codon

• Silent mutation - does not change the amino acid

• frameshift - changes the reading frame

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Effects:

• silent

• loss of function

• gain of function - overactive or cannot be turned off

• conditional - some enzymes only active under certain conditions, mutation can change these conditions

What is a disease resulting from a missense mutation (point mutation)?

Sickle Cell Anaemia

• A→T

• Glu→Val

• results in clumped haemoglobin, instead of globular

• affects shape of red blood cells

• high risk of blood clotting

What is a disease resulting from a nonsense mutation (point mutation)?

Dopa-responsive dystonia and depiapterin reductase 

• A→T

• Lys→stop

• affects muscle contraction (stiffness, tremors, coordination etc.)

What is a disease resulting from a deletion/ insertion mutation (point mutation)?

A little info: e.g. can result from a depurinated A base

• Wild type - results in premature stop codon

• Frameshift - results in a change of reading frame

Disease: Duchenne (DMD) and Becker Muscular Dystrophies (BMD)

• progressive muscle wasting due to mutations in 79-exon

• Dystrophin connects muscle cytoskeleton to ECM

Duchenne is much more severe than Becker

• Duchenne - frameshift mutation that often results in premature stop codon

• Becker - 3 codon in frame deletion

Large-Scale mutations:

occur at chromosomal level

Duplication:

• large sections of chromosome is duplicated 

• often occur during replication

Inversion

• a segment of chromosome is inverted

Deletions:

• large scale deletions 

Insertions:

• Large scale insertions

Translocation:

• swap of DNA between 2 different chromosomes 

What is a disease resulting from a deletion mutation (Large Scale)?

DiGeorge Syndrome 

• deletion in part of chromosome 22

• delayed development

• congenital heart defects

• reduced immune function 

• cleft palate

What is a disease resulting from a translocation mutation (Large Scale)?

Burkitt’s Lymphoma

• translocation from chromosome 8

• results in increased activity of c-myc (transcription facto involved in regulation of proliferative gene)

• results in lymph node tumours

What causes mutations?

Spontaneous - occur naturally

• arise in all cells at low frequency

• error sin DNA replication

• spontaneous lesions/ damage

Induced - require a mutagen

• chemical (e.g. base analogues)

• radiation (e.g. UV light)

Spontaneous mutations:

Depurination

• purine bases can be lost from the sequence

Deamination:

• deamination of cytosine → Uracil

Tautomeric forms of DNA bases:

• bases can be incorporated inro DNA in their rare tautomeric forms

• allows different base pairing

Cystic Fibrosis:

Different CFTR mutations have different consequences on the protein

• CFTR encodes for cystic fibrosis transmembrane conductance regulator (a protein that sits within the cell membrane and is a chloride channel - important for epithelial fluid transport between cell and external environment)

• cystic fibrosis results in dysregulation of epithelial fluid transport (prevents fluid from leaving the cell, resulting in buildup inside the cell)

What is human variation?

• humans have variation in their genes

• arise from mutations (e.g. ACE gene II, ID, DD)

• majority of differences occur in non-coding regions of our DNA

Are mutations bad?

Can be bad (disease) or bad (evolution)

Mutations in evolution:

Good mutations give us a selective advantage (advantageous trait which helps us thrive)

E.g. high altitude adaptation: animals and people who lived in high altitudes had bigger chests and greater lung capacity

E.g. CCR5 receptor helps HIV-1 enter cells

• people who carry a particular mutation in the CCR5 receptor are protected against HIV-1 (32 bp deletion on CCR5 gene)

• reduces amount of receptor on immune cell (which HIV binds to)

What are neutral mutations?

Neither advantageous nor disadvantageous

• don’t actually affect the protein (silent mutation)

• majority of mutations are neutral

• can also spread in a population

• can be useful for forensic testing and paternity tests

Disease mutations:

Why do many disease-causing mutations persist in the population?

Many genetic diseases require 2 inherited mutated copies of the same gene 

• recessive inheritance

• but many people are heterozygous (carriers of the disease without presenting symptoms) 

• if mutation confers some selective advantage to heterozygous people, it may be maintained in the population by natural selection 

E.g. Sickle cell anemia

• heterozygous people (Aa) have one good copy (so have enough red blood cells so they only have mild symptoms sickle cell. But they have protection from malaria. 

How can new genes/ DNA be introduced into a genome?

1. mutation - particularly if this mutation presents with some advantage

2. duplication - can happen during replication

3. DNA segment shuffling - can occur during replication (translocations happening between the genes)

4. horizontal gene transfer - transformation, conjugation and transduction (bacterial cells)

Gene families:

Genes that are related

E.g. Globin family

• consists of both an alpha and beta version of the globin protein and gene. This forms haemoglobin.

• these related proteins all evolved from the same original ancestral gene

• results of an initial gene duplication, translocation and then subsequent mutations

What is the effect of mutation/ inactivation of DNA repair genes?

• increase rate of mutation 

• because there is no longer the proteins to repair the DNA

What are some mechanisms to prevent replication errors?

Proofreading polymerase (fixing majority of errors)

• errors occur usually in 1:100,000 to 1:1,000,000 bases

• with proofreading 1:100,000,000 bases

• fixes up to 99% of errors

• 3’-5’ exonuclease activity of the polymerase removes several bases (including the incorrect one) → then replication resumes in 5’ to 3’ direction

• occurs during S phase (replication)

Mismatch repair system

• mismatch repair enzyme recognises and removes/ replaces the nucleotide

• identifies errors in the secondary structure (e.g. tautomeric bases)

• mismatch repair enzymes recognize this and bind to base (MutS)

• MutL scan DNA to find nick

• region between mismatch and nick will be removed by endonucleases and DNA polymerase fills in the gap

• occurs mostly in S phase 

DNA damage: What are some types of DNA damage that can occur?

Oxidative damage:

• guanine is more susceptible

Hydrolytic attack:

• cleave chemical bonds in DNA

• can result in removal of a base or deamination

Uncontrolled methylation:

• alkylation of bases

• can change the base pairing

DNA damage by hydrolysis (hydrolytic attack):

• repairable damage

Depurination = spontaneous loss of purine bases (adenine and guanine) by hydrolysis

• backbone remains intact but base is lost

• can result in a deletion (frameshift)

• after several rounds of replication, this mutation becomes incorporated into daughter cells

Deamination = spontaneous conversion of a cytosine to uracil (which have different base pairing) by hydrolysis

• mismatch occurs (no longer pairs with guanine)

DNA damage by alkylation/ methylation:

• Guanine particularly susceptible (alkyling event → methyl group attached to oxygen atom on guanine)

• affects base pairing (methyl guanine pairs with thymine, not cytosine)

• result in base substitution

DNA damage by UV Irradiation: 

Covalent linkage between two adjacent pyrimidine bases 

• caused by UVB radiation from the sun 

• Thymine dimers: covalent linkages on the C-C bonds from lesions 

• can occur between any two neighboring pyrimidine bases (T or C)

• if left unrepaired, end up with permanent damage 

• e.g. melanoma 

• can be repaired (when one strand is damaged, the complementary strand remains intact and can be used to restore correct nucleotides to damaged strands)

DNA damage repair pathways: Base lesions - single or double

Base excision repair (BER)

Nucleotide excision repair (NER)

• for both of these, damage is excised (physically excise and remove damaged nucleotide/ string or nucleotides). Original sequence then restored using undamaged complementary strand.

• Enzymes involved: DNA polymerase (to restore) and DNA ligase (to seal them up)

Direct reversal repair (DR)

• Direct removal of lesion

• no cleavage or ligation

Base Excision Repair (BER):

What is it?

What events does it respond to (what types of mutation)?

What are the enzymes involved?

Repairs damage to a single base

• occurs when an event like deamination or depurination has occurred (damage to the base rather than whole nucleotide)

A set of enzymes acting sequentially:

DNA glycosylases:

• scan DNA and identify DNA to single bases (such as deamination or depurination) 

• recognise specific type of altered base by ‘flipping out’ from helix 

• excise/ remove base via hydrolysis (breaking bonds attaching the base - does not break backbone)

AP endonucleases (AP = apurinic or apyrimidinic):

• recognise the phosphodiester bond which is missing a base

• cut the phosphodiester backbone

DNA polymerase: 

• adds a new nuclotide

DNA ligase:

• seals up the nick

Nucleotide Excision Repair (NER):

Repairs larger changes to DNA helix: 2 or more bases damaged

• entire nucleotides removed

• can be used to repair pyrimidine dimers (induced by UV radiation)

What are the enzymes involved:

Multicomplex enzyme (scans for DNA distortion)

• several enzymes apart of this multicomplex enzyme

• this complex cleaves the phosphodiester backbone of abnormal strand on both sides of distortion 

DNA helicase:

• unwinds the region of DNA 

• stretch of oligonucleotides removed from either side of lesion (a few extra bases also removed)

DNA polymerase:

• fills gaps

DNA ligase:

• seals

Example of disease:

Xeroderma pigmentosum 

• recessive genetic defect

• enzymes required for nucleotide excision repair are mutated

• makes them very sensitive to UV (sunlight)

• high risk of developing skin cancer 

Transcription coupled DNA repair: 

• Occurs during transcription

• Ensures cell’s most important DNA is efficiently repaired 

• Links excision repair system with RNA polymerase 

• RNA polymerase stalls at DNA lesions and directs repair machinery to these sites

• targets repair to genes that are actively being transcribed into mRNA (making it efficient) 

Disease where this doesn’t work:

Cockayne syndrome

• recessive congenital disorder

• the RNA polymerase that would stall lesion is permanently stalled/ unable to return to polymerization = high level of apoptosis (programmed cell death) = more cell death

Direct Reversal Repair (DR): 

Most efficient form of DNA repair

• rapid removal of certain highly mutagenic or cytotoxic lesions (e.g. alkylation lesion 6-O-methylguanine) 

• does not require any removal of bases or nucleotides

• used to repair alkylation/ methylation 

Enzyme involved:

methyltransferase (MTase) protein:

• accepts methyl group on cysteine residue from alkylated guanine nucleotide.

• This corrects chance of pairing with wrong base

• restores normal guanine

• MTase then inactivated 

• No DNA cleavage or ligation 

Emergency repair of heavily damaged DNA:

Regular DNA polymerase (Pol III) stalls when it encounters DNA damage and release DNA (highly accurate)

In emergencies, they employ less accurate back-up polymerases to replicate through the DNA damage - translesion polymerase (Pol V) 

• can continue polymerisation despite DNA damage

• doesn’t have proofreading activity (instead just keeps going)

• but it falls off (doesn’t bound that long) then Pol III continues. 

• Risky for cell as it incorporates mutations 

• not preferred form of repair (not even really repair just allows replication to continue)

When does DNA repiar occur?

Proofreading and mismatch occurs during S phase (DNA replication)

Checkpoints:

• M phase Checkpoint

• G1/S phase checkpoint:

• G2/M phase checkpoint:

• Intra- S-phase checkpoint:

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Cell cycle stops if damaged DNA is detected

In mammalian cells, the presence of DNA damage can:

• block entry from G1 to S phase (checkpoint)

• slow S phase (replication) once it has begun

• block transition from G2 phase to M phase (checkpoint)

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Some proteins involved in regulating the cell cycle:

ATM protein:

• large kinase (phosphorylates proteins) that signals intracellularly to delay the cell cycle in response to DNA damage

• Individuals with ataxia telangiectasia (AT) (defects in ATM protein) suffer from effects of unrepaired DNA lesions (neurodegeneration, genome instability etc)

p53: ‘Guardian of the genome’

• arrests the cell cycle at G1/S checkpoint until damage is repaired

• activates DNA repair enzymes

• can initiate apoptosis is damage is too great (if arrested too long)

• huge implication in cancer - mutations in P53 can prevent apoptosis = cancer

CHK1: Kinase

• cycle arrest at S and G2/M checkpoints 

• DNA repair or cell death