Topic V: Recombination, Repair and Mutations W10-12

What are the different types of mutations?

*Point mutation

• Replace on base with another

• Leads to a change in one aa

*Frame shift

• Add/delete bases

• Changes to reading frame

• Changes more than one aa

• More significant effects

^Non-sense mutation

• RNA sequence becomes a stop codon

• Very bad - more significant

^Missense mutation

• Changes amino acids from one to another

^Silent mutation

• No affect on the protein (due to redundant nature of aa)

^Conservative mutations

• Produces amino acids of the same nature

• E.g. Glu mutates to an Asp but they are both acidic

^Non-conservative

• Produces amino acids of different nature from original

• E.g. Ser (polar) replaces with Phe (non-polar)

*Named after how they affect the DNA

^Named after how they affect the protein

What are the types of DNA damage?

• Double-stranded break

  • Caused by cellular activity, ionising radiation, chemotherapeutic drugs, DNA repair of other types of damage

  • We can potentially loose a part of the chromosome

• Chemical bound between neighbouring nucleotides

  • Can be caused by UV light

  • This is INTRAstrand crosslinking between nucleotides (aka the same strand)

  • Repair mechanism: Nucleotide Excision Repair

• Chemical linkage of two strands

  • Can be caused by reactive oxygen species, chemotherapeutic drugs or other cellular and environmental chemicals

  • INTERstrand crosslinking between strands

  • Repair mechanism: Homologous Recombination

• Chemical modification of a nucleotide

  • Can be caused by reactive oxygen species, chemotherapeutic drugs or other cellular and environmental chemical, as well as normal modification that regulate what genes are active

  • These occur very often but the cell if very good at repairing this

  • Methylation, Depurination and Deamination

What are the way that DNA can repair itself?

Direct repair

• Fix DNA molecules that carry nucleotides damaged by mutagens

• Find a DNA that has been changed, place that change onto an enzyme

• Search/fix abnormal chemical structures in DNA

Excision repair

• Fix DNA molecules that carry nucleotides damaged by mutagens

• Damaged base/ is excised from a nucleotide by a specific DNA glycosylase and phosphodiesterase and then fixed via a DNA polymerase and sealed by a DNA ligase

• Search/fix abnormal chemical structures in DNA

Mismatch repair

• Fix mismatched but otherwise normal nucleotides that result from errors in replication

• Detects absence of proper base pairing between parent and daughter strands (MutS/MutH)

Nonhomologous end joining

• Use to mend double-strand breaks in DNA

• Ku binds to each end and helps bring the 2 fragments close together and then they are then ligase together, but this is hard to do

• Very low fidelity - very common to errors

What are inherited (above) and non-inherited mutations?

Inherited (germline mutations)

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

• Acquired from parents (Predisposition (familial traits))

• May be transmitted to offspring

• Responsible for single gene disorders: cystic fibrosis

• May or may not be present in multifactorial disorders: cancer, heart disease, diabetes, Alzheimer’s (aka diseases caused by multiple mutations)

Non-inherited (somatic mutations)

• Affect only the mutant somatic cell and its descendants

• Will not be transmitted to offspring

• Could occur due to environmental factors and build up over time (at the start we can fight back and fix these mutations but over time as these mutations accumulate we can no longer deal with them and the consequences of the disease can present (e.g. cancer))

• Contribute to multifactorial disorders: cancer, heart disease, diabetes, Alzheimer’s etc. (sporadic cases, also act as triggers for those with pre-disposition)

• Cumulative → Accumulate in age related processes, UV and mutagen exposure...

What are the major classes of mutations?

• One impact includes the grain of function, this is where a mutation causes over activity in the mutated cell, this is the case with cancers, where mutations lead to overgrowth and over proliferation of tumours etc.

• Conditional → certain enzymes are only active under specific conditions, these mutations changes those conditions that enzymes act in

Point mutations

• Single base substitution (missense, nonsense, silent) or single base deletion/insertion (frame shift)

• Base substitution → no change in the overall number of bases

  • Missense → Results in an alternate amino acid (e.g. sickle cell)

    • This can be very impactful depending on the location of mutation (e.g. active site)

  • Nonsense → Results in a premature stop codon (e.g. Dopa-responsive dystonia and Sepiapterin reductase

    deficiency)

    • If this mutation occurs very downstream of the active site (tail end) then it may not necessarily impact function

  • Silent → Does not affect the Amino Acid Sequence (due to redundant nature of aa)

• Base deletion/insertion

  • Results in a changes in the number of bases (e.g. Duchenne (DMD) and Becker Muscular Dystrophies (BMD))

**If a base is substituted and results in a STOP codon → Missense

If a base is inserted/removed and results in a STOP codon → Frame shift

Sickle Cell Anaemia case

• Sickle Cell Anaemia → Point mutation results in clumped haemoglobin, instead of globular

• Gene: HBB encoding beta globin protein

• Associated Disease: Sickle Cell Anaemia

• Mutation: Point mutation – base substitution: A>T

• Effect on translated protein: Missense Glu>Val

• Symptoms

  • Fatigue and anaemia

  • Bacterial infections

  • Sudden pooling of blood in the spleen and liver congestion

  • Lung and heart injury

  • Leg ulcers

Dopa-responsive dystonia and Sepiapterin reductase deficiency

• Gene: sepiapterin reductase gene (SPR)

• Associated Diseases: Dopa-responsive dystonia and Sepiapterin reductase deficiency

• Mutation: Point mutation – base substitution (A>T)

• Effect on translated protein: premature stop – nonsense (Lys>Stop)

• Sepiapterin reductase is an enzyme required for the synthesis of key neurotransmitters dopamine and serotonin

• Deficiency: Symptoms include involuntary muscle contractions (dystonia), muscle stiffness, tremors, problems with coordination and balance (ataxia), and involuntary jerking movements

Duchenne (DMD) and Becker Muscular Dystrophies (BMD)

• Gene: dystrophin

• Associated Diseases: Duchenne (DMD) and Becker Muscular Dystrophies (BMD)

  • DMD: severe, BMD: mild

• Progressive muscle wasting diseases due to mutations in the 79-exon dystrophin gene

• Dystrophin connects the muscle cytoskeleton to the ECM

• Duchenne muscular dystrophy (DMD)

  • Often results in frameshift or Nonsense mutation: Truncated protein

• Becker Muscular dystrophy (BMD)

  • In-frame mutation (deletion) (entire codons removed i.e. nucleotide multiple of 3 are removed)

  • Does not affect the frame downstream: smaller protein but retains some function and very similar to the wild type

What are some larger scale mutations?

• DID IT (acronym)

• This occur at chromosomal level and affects MULTIPLE GENES

• very important for evolution

• Deletion

  • DiGeorge syndrome, large scale deletion of part of chromosome 22

  • Delayed development, congenital heart defects, reduced immune function, cleft palate

• Translocation (image above)

  • Burkitt’s Lymphoma, translocation from chromosome 8

  • Results in increased activity of c-myc (proto-oncogene)

    • A proto-oncogene can the potential of becoming an oncogene which leads to cancer

  • Transcription factor involved in regulation of proliferative genes

  • Results in lymph node tumours (cancer)

  • Cause? → commonly associated with Epstein-Barr Virus

What causes mutations?

• Spontaneous - occur naturally

  • Arise in all cells at low frequency

  • Errors in DNA replication (e.g. due to tautomers)

  • Spontaneous lesions or damage (e.g. depurination, deamination of cytosine) (image)

    • Sites on each nucleotide known to be modified by spontaneous: (below)

    • Oxidative damage (red arrows): METABOLIC guanine is more susceptible

    • Hydrolytic attack (blue): CLEAVES chemical bonds in DNA (this causes a removal of a base or deamination)

    • Uncontrolled methylation: (green): ALKYLATION of bases (this changes the base pairing)

• Induced - require a ‘mutagen’

  • Chemical → Base analogues, intercalating agents, base modifiers

    • E.g. Aflatoxins, benzopyrenes, nitrites

  • Radiation → UV light, electromagnetic ionizing agents (free radicals)

    • UVB damage causes pyrimidine dimers that results in a lesion or a kink in the DNA this can lead to incorrect replication/base pairing thus can lead to a permanent mutation (e.g. melanoma)

• Inherited - also known as germline

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

  • Acquired from parents

  • May be transmitted to offspring

Induced DNA damage: 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 form lesions

• Can occur between any two neighbouring pyrimidine bases – (T or C)

• UV irradiation leads to: Sunburn, increased melanin production, If left unrepaired: Can lead to melanoma (cancer)

Spontaneous DNA damage by hydrolysis (damage to the bonds)

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

  • Depurination leads to loss of a nucleotide pair (deletion)

    • The backbone remains intact but the base is lost

  • When replication machinery encounters missing purine on template, it skips to next nucleotide resulting in a deletion: frameshift

  • Can be repaired well

• Deamination = spontaneous conversion of cytosine to uracil by hydrolysis

  • Deaminated cytosine becomes uracil and mutation propagated as uracil pairs with adenine: base substitution

    • As a result of a hydrolytic event the amine group of a cytosine is cleaved, which then converts the base to uracil (the base still remain intact, its just the base changes)

  • If deamination remains we end up in a change to the complementary base and a mutation will be introduced into the 2nd strand that will then be incorporated into the genome following replication of that

*Note that one copy of the DNA remains unchanged as these mutations only occur to one strand, during replication both the normal and mutated strands separate and are replicated

Alkylation Damage: Methylated Guanine

• Guanine is particularly susceptible to this type of damage

• Results in an altered base that doesn’t follow base pairing rules

• Alteration to the base – Methyl group attached to oxygen of guanine

• Methyl Guanine pairs with thymine, not cytosine – Base substitution

Spontaneous mutations - Tautomeric forms of DNA bases

• Bases can be incorporated into DNA during replication in their rare tautomeric forms

• Base pairing will follow different rules → Base substitution

What are the effects of mutations within genes?

• Reduced quantity and efficiency and prevention of protein forming would all be a loss of function mutations

• Change in proteins function → could be a gain/loss of function

What are CFTR Mutations?

• Large gene with many known mutations

  • Not all these mutations will cause CF, some can go unnoticed

  • So many people can have different CFTR genes but not necessarily present with actual CF

• CFTR encodes for the Cystic Fibrosis Transmembrane Conductance Regulator

  • Membrane protein

  • Chloride channel

  • Very important for epithelium fluid transport between the cell and the cytosol

• Cystic Fibrosis: dysregulation of epithelial fluid transport

• F508del results in low numbers of misfolded protein in the cell membrane (this is an example of the very common deletion mutation)

Complex mutations: CFTR gene associated with cystic fibrosis in humans

• Large variation in the CFTG gene in the population

• Remember not all of them lead to CF (come can be silent/small)

What is the Human Variation in the genes?

• We are different in large part because the sequence of our DNA is different

  • On average <1 difference in every 1,000 bp

  • This is still ~ 1 x 10⁶ differences between individuals

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

• However, even amongst our 25,000 or so genes, at least one third are polymorphic within the healthy population

• Each different form of a gene is called an allele and has arisen as a result of mutation

Mutation in Evolution: Principle

• Some mutations that provide a selective advantage are perpetuated: natural selection

  • These mutations give a higher chance of surviving and thus can be passed one (i.e. selected)

• High Altitude adaptation: Tibetans and Nepalese live altitude 4,000m

  • Air contains 40% less oxygen

  • Compensate with bigger chests: greater lung capacity

  • Evolution of few red blood cells with better blood flow in brains

  • These mutations have lead them to thrive in these environments

Example: CCR5 receptor helps HIV-1 enter cells

• CCR5 receptor is a type of chemokine receptor present on immune cells and is what HIV particles will bind to in order to infect an individual

• Therefore people who have the mutation where they do not have those receptors are protected against HIV

• Beneficial mutation up to 14% in Northern Europe

• So we see that the 32 allele is common where HIV is rare and 32 allele is rare/absent where HIV is rampant

What are Neutral Mutations?

• Neither advantageous nor disadvantageous

• They don’t cause disease but they also do not provide any evolutionary advantage because they don’t actually affect the proteins or any coding regions of DNA (aka silent mutation), but they are very scientifically useful in forensics, paternity testing etc.

• The majority of mutations are neutral and inherited

• Can also spread in population

• In humans in the form of single-nucleotide polymorphisms (SNPs)

  • 99.5% similarity

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

• There are a multitude of conditions/diseases (> 4000) which result from the ‘bad’ or detrimental effects of mutations that are passed on through the generations → so why do they persist in the population

• Many genetic diseases only manifest when the individual has inherited 2 mutated copies of the same gene (homozygous)

  • Recessive inheritance: the affected person must be homozygous for a disease mutation or compound heterozygous (mean 2 faults genes) for two different disease-causing mutations for the condition to manifest

  • No working copy of the gene to provide sufficient function

• In these cases, heterozygotes with one normal gene copy and one mutant gene copy are healthy (most of population?)

  • Their normal gene provides sufficient normal protein/function to compensate for this mutated allele → so disease does no present

• Heterozygote Advantage: If the mutation confers some selective advantage to heterozygous individuals while not causing diease, it may be maintained in the population by natural selection (image)

  • E.g. Homozygous to CF causes disease but being a heterozygote causes protection against cholera

Sickle Cell Allele and Malaria

New genes and genome evolution

• DNA is not made in random sequences, it is generated from pre-existing DNA

• Changes in DNA sequence lead to changes in structure and the development of new functions for proteins

• Intragenic mutation

  • Could be simple point mutations, deletions, insertions

  • So the original gene picks up mutations and begins to evolve, particularly if the particular mutation is advantageous

  • So new genes are created through mutation

• Gene duplication

  • New genes introduced via duplication

  • This can happen during replication

  • We then have 2 copies of the gene that can evolve at different rates (e.g. they can acquire mutations etc. at different locations at different rates) and essentially become completely different genes

• DNA segment shuffling

  • Happens during replication

  • This is where translocations and stuff occurs (during recombination)

  • This can cause new combinations of genes

• Gene mixing/ Horizontal gene transfer

  • This uptake can occur from: Bacterial transformation, Conjugation, Transduction, Viral reassortment (above)

Evolution through expansion of Gene Families

• These DNA duplications, mutations and shuffling then give rise to different gene families

• These genes that are related are called gene families

• Example

  • The alpha and beta subunits are only slightly different, and thus comes from an initial duplication, translocation and then subsequent mutations of the ancestral gene

• Genes evolve as families: members encode proteins with related structure and function

• Globin gene family:

  • Derived from common ancestral gene

  • Encode globular proteins transporting oxygen

  • Common to many species

  • Essential for multicellular animals to grow to large size

  • Occurred over millions of years

  • At 400 million years there was a translocation event where the alpha section split off from chromosome 11 and translocated onto chromosome 6

*Note that not all genes evolve at the same rate

Why is Genetic Stability important?

• For survival, organisms not only need accurate mechanisms for replicating DNA but also mechanisms for repairing spontaneously occurring damage in DNA

• We do not want long lasting permanent mutation occurring that can cause ongoing or permanent damage to our proteins and subsequently cause disease

• DNA damage can be caused by heat, metabolic accidents, radiation, exposure to the environment, different substances

• Fewer than one in 1000 accidental base changes results in permanent mutation thanks to DNA repair

• This is very important for genetic stability, if our cells are acquiring mutations at a really high rate then they are not going to be able to replicate successfully

Why is DNA repair critical?

• DNA repair occurs through a range of coding genes involved in repair

• Inactivation of DNA repair genes causes an increased rate of mutation (as other mutations start to build up) → this can lead to disease

• Many of these were originally identified in bacteria

• Many serious human diseases are linked to decreased DNA repair enzymes/genes

  • Cancers, UV sensitivity, leukaemia, growth and development

Recap from Theme I: Errors during replication

• If errors not fixed during replication: mutations arise

• Errors such as:

  • Tautomeric bases

  • Mismatch

• 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

• Mismatch repair system

  • Identifies errors in the secondary structure

  • Mismatch repair enzymes recognize this and remove/replace the nucleotide

Recap from Theme I: Proofreading Polymerase

• If errors not fixed during replication: mutations arise

• Proofreading polymerase (fixes majority of errors: up to 99%)

• Incorrect base paired, elongation pauses

• 3’-5’ exonuclease activity of the polymerase removes several bases, including the incorrect one

• Replication resumes 5’-3’

• Occurs during replication (S phase)

Recap from Theme I: Mismatch Repair

• If errors not fixed during replication: mutations arise

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

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

• MutL scans downstream of the DNA to find a nick

• Region between mismatched base and nick excised by exonucleases

• DNA polymerase fills the gap and DNA backbone sealed via DNA ligase

• Occurs mostly in S phase of the cell cycle, follows behind replication

How can the DNA double helix be repaired?

• We have two separate copies of all genetic information (double helical structure of DNA)

• When one strand is damaged, the complementary strand (copy of same information) remains intact

• This is used to restore correct nucleotides to damaged strand

Overview of DNA damage repair pathways

• Base lesions: single (methylation, depurination) or double (pyrimidine dimers)

• Base excision repair (BER)

• Nucleotide excision repair (NER)

• For BER & NER

  • Damage is excised (removed)

  • Original sequence restored by using undamaged strand as template

  • Remaining break sealed with ligase

• Direct reversal repair (DR)

  • Direct removal of lesion

  • No cleavage or ligation of a base, instead we remove the lesion (e.g. methylation - we remove the methyl group)

Base Excision Repair

• Repairs damage to a single base (deamination or depurination - damage to the base as opposed to the entire nucleotide)

• A set of enzymes acting sequentially

• Specific DNA glycosylases

  • Recognise specific type of altered base by ‘flipping out’ (rotate) from helix

  • Excise/remove base via hydrolysis (breaking the bonds b/w sugar and base NOT the phosphate backbone)

• AP endonucleases (AP for apurinic or apyrimidinic)

  • Recognise this phosphodiester backbone with a missing base in helix

  • Then cuts the phosphodiester backbone

• DNA polymerase adds new nucleotides into the gap

• DNA ligase seals the nick

Nucleotide Excision Repair

• Repairs larger changes to DNA helix: 2 or more bases and entire nucleotides need to be removed

• Multi-enzyme complex scans DNA for distortion

• This complex will then cleaves phosphodiester backbone of abnormal strand on both sides of distortion

• DNA helicase (DNA unwinding enzyme) peels away single-stranded oligonucleotide containing lesion

• Gap filled by DNA polymerase

• Sealed with DNA ligase

Xeroderma pigmentosum - Nucleotide Excision Repair

• Autosomal recessive genetic defect: nucleotide excision repair enzymes are mutated (inherited)

• Prevalence 1 in 250,000

• Symptoms: severe sunburn after minutes of sun exposure, freckle

• UV light can cause mutations and without proper nucleotide excision repair mechanisms due to accumulation of unrepaired DNA

• High risk of developing skin cancers: tumour suppressor genes are affected, melanoma

• Life expectancy shorter by ~30 years

Transcription-coupled DNA repair

• Occurs during transcription

• Ensures cell’s most important DNA is efficiently repaired

• Links excision repair systems with RNA polymerase (enzyme that transcribes DNA into RNA)

• RNA polymerase stalls at DNA lesions and falls off at these points and this then directs the nucleotide excision proteins (DNA glycosylases and AP endonucleases) to these sites

• Targets repair to genes that are actively being transcribed into mRNA

Cockayne syndrome - Transcription-coupled repair and human disease

• The particular RNA polymerase that could stall or detect the lesion is permanently stalled or is unable to return to polymerisation and this then results in a high level of apoptosis as they encounter high levels of DNA damage as cells age

• Autosomal recessive congenital disorder with a high level of apoptosis

• Prevalence: 1 in 200,000

• Symptoms: growth retardation, skeletal abnormalities, progressive neural degeneration and retardation, severe sensitivity to sunlight

• Defect in transcription-coupled repair

• RNA polymerase molecules become permanently stalled at sites of DNA damage in important genes

• Causes cell apoptosis (programmed cell death)

• Life expectancy 10-20 years

Direct Reversal Repair

• Most efficient form of DNA repair

  • As it does not require the removal of bases or nucleotides

• Rapid removal of certain highly mutagenic or cytotoxic lesions

  • E.g. Alkylation lesion 6-O-methylguanine

• Methyltransferase (MTase) protein accepts methyl group (CH3) on cysteine residue from alkylated guanine nucleotide

  • Restores normal guanine

  • MTase inactivated

  • No DNA cleavage or ligation required

Emergency repair of heavily damaged DNA

• Highly accurate replicative DNA polymerase (Pol III) stalls and falls of when it encounters damaged DNA

• In emergencies, they employ less accurate back-up polymerases to replicate through the DNA damage - translesion polymerases (Pol V) from where the Pol III has left off

  • Pol V is able to replicate DNA and continue polymerisation despite damage being identified (usually they be falling off/stalling), hence transcribed through the lesion thus called translesion polymerase

• The back-up polymerases lack exonucleolytic proofreading activity

• These polymerases only add one or a few nucleotides before it falls off and the replicative polymerase (Pol III) continues from there

• Risky for the cell: responsible for many mutations as the mutations become incorporated into the DNA

DNA damage can delay progression of cell cycle

• When does repair occur?

• In most cells, DNA damage causes a delay in cell cycle

• Ensure that all damaged is repaired before a cell divides

  • G2/M checkpoint → does not enter mitosis until this is happed

  • Intra S phase checkpoint → S phase is slowed down when DNA damage is detected

Cell Cycle involves critical check points

• Cell cycle will not progress pass these check points until damage is repaired

• Orderly progression of cell cycle maintained through use of checkpoints to ensure completion of one step before next step begins

• 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)

• Delays facilitate DNA repair by providing time needed for repair to reach completion

Integrating DNA repair and cell cycle progression

• DNA damage results in increased synthesis of some DNA repair enzymes

• Special signalling mechanisms that arrest the cell cycle and respond to DNA damage

• ATM protein: large kinase (phosphorylate 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 checkpoints until damage repaired

  • Activates DNA repair enzymes

  • Can initiate apoptosis if damage too great (if cell is held at rest for too long)

  • Huge implication in cancer: tumour suppressor

• Chk1: kinase

  • Cycle arrest at S and G2/M checkpoints

  • DNA repair or cell death

Mitosis Revision

1. Prophase: chromosomes condense and become visible, the nuclear envelope breaks down, and spindle fibers form.

2. Metaphase: chromosomes line up along the middle of the cell (the equator).

3. Anaphase: sister chromatids are pulled apart to opposite poles.

4. Telophase: nuclear envelopes re-form around each set of chromosomes, and the chromosomes uncoil.

Chromosomal Crossover (homologous cross over) Revision

• When: Prophase I of meiosis

• What happens: Homologous chromosomes (one from each parent) pair up as a tetrad, which is 4 chromatids in total. Non-sister chromatids break and swap matching sections

• Chiasma (plural: chiasmata): the point where the swap happens

• Result: Each chromosome ends up with a mix of maternal and paternal DNA. The genes are the same, but the alleles (variants) can differ

• Why it matters: It creates genetic variation in gametes, on top of the variation from different sperm meeting different eggs

Meiosis Revision

• Before it starts: Interphase, where DNA replicates, so each chromosome has 2 sister chromatids

  Meiosis I (homologous pairs separate, so the cell becomes haploid)

  1. Prophase I: Chromosomes condense and the nuclear envelope breaks down. Homologous chromosomes pair up as tetrads, and crossover occurs.

  2. Metaphase I: Homologous pairs line up at the middle in random orientation (independent assortment).

  3. Anaphase I: Homologous chromosomes are pulled to opposite poles. Sister chromatids stay together.

  4. Telophase I and cytokinesis: The cell splits into 2 haploid cells, each with chromosomes that still have 2 chromatids

• Meiosis II (sister chromatids separate, similar to mitosis)

  1. Prophase II: The spindle forms in each of the 2 cells, and there is no DNA replication.

  2. Metaphase II: Chromosomes line up at the middle.

  3. Anaphase II: Sister chromatids are pulled apart.

  4. Telophase II and cytokinesis: Each cell splits again

• Result: 4 genetically unique haploid cells (gametes).

  Key contrast: Meiosis I separates homologous chromosomes, and meiosis II separates sister chromatids.

Humoral response

• The humoral response is commonly activated to fight bacterial infections or toxins, and relies on using antibodies to destroy the foreign antigens

• The response is tightly regulated, and involves several steps of clonal selection: specific cells are selected and cloned to fight a specific target

• Cells that are clonally selected will then clonally select other immune cells, creating a rapid expansion in the number of lymphocytes targeted to a specific pathogen

• This rapid expansion of lymphocytes is one reason an infected person may feel fatigue.

What are the function of antibodies?

• Antibodies are small Y shaped proteins that are secreted by Plasma B cells

• They destroy or deactivate the pathogen when they bind to it

• They are highly specific to a particular antigen

• The level of antibodies in your blood can indicate the strength of your immunity to that specific pathogen

Diagram

• Antibody

  • All antibodies have a constant region (a region that is the same for all antibodies). However, there is a region at the two tips of the antibody that can be different, called the variable region

• Agglutination is when pathogens become trapped in a network of antibodies, making it easier for them to be phagocytosed (engulfed) by other lymphocytes, for example macrophages

• Antibodies can also bind to toxins that are either produced by a pathogen, or directly introduced into a person (e.g. through a snake bite). In neutralisation, the antibodies deactivate these toxins by binding to them, thereby stopping these toxins from affecting other cells

• The final way antibodies function is through boosting the innate response of opsonisation. Antibodies coat the pathogen’s surface to ‘tag’ the pathogen. This makes it easier for immune cells to identify the pathogen, and also prevents the pathogen from binding to host tissues and helps phagocytes to phagocytose them

• Complement activation is a process where antibodies, when bound to a pathogen, trigger a cascade of other proteins to be activated. These complement proteins will have other functions, e.g. physically ‘punching’ a hole in the pathogen’s membrane, making a pathogen more easily identified, or deactivating a toxin. Complement proteins work as part of the innate immune response and while their action is boosted by working with antibodies, complement proteins can work independently of antibodies

What is the Cell-mediated response

• It is commonly activated to fight cancerous cells or cells that have been infected by viruses

• The cell-mediated response relies on using cells (e.g. cytotoxic T cells) to destroy other diseased cells, hence the name ‘cell-mediated’

  • This differs from the humoral response that uses antibodies to fight an infection

• Like the humoral response, the cell-mediated response involves a cascade of cells being activated and dividing. This large consumption of resources contributes to the symptoms a sick person may feel, such as fatigue.

Diagram

• If a cell becomes diseased, for example cancerous or infected by a virus, it will present this information on the MHC I class protein, beginning the cell-mediated immune response (Tip: MHC I class proteins are for endogenous antigen)

• Antigen presenting cells such as dendritic cells or macrophages patrol through the body’s tissues. If one encounters a foreign antigen from a diseased cell, it will phagocytose it and then present a fragment, or antigen on its MHC II class protein. It will then travel to the lymph node via the lymphatic system to get back-up

• When an antigen presenting cell arrives in the lymph node, it will activate a specific T cell by presenting the antigen fragment on a MHC II class protein. This process is called selection

• The selected T cell will then begin to divide into memory T cells and helper T cells which subsequently differentiate into either effector or memory T cells. This process is called clonal selection

Overview of the Adaptive immune response

What are Double stranded breaks?

• Both strands are broken

• Problem: no template for repair

• Causes:

  • Environmental damage:

    • Double stranded breaks can occur from ionising radiation (e.g. x-rays) and reactive chemicals (e.g. anti tumour-agents)

  • DNA replication:

    • Most of the time they arise from DNA replication forks that become stalled or broken (accidents occur during nearly every round of DNA replication)

• If lesions not repaired:

  • Breakdown of chromosome into smaller fragments

  • Catastrophic loss of genes when cell divides

Double stranded break during replication

• These unrepaired lesions could be a result of a deamination or depurination → can be fixed via emergency repair

• These unrepaired breaks leads to a double stranded break

What are the two mechanisms of Double stranded break repair

• Non-Homologous End Joining (NHEJ)

  • Re-joining of broken ends by DNA ligase'

  • Generally loss of nucleotides at site of joining

  • Common in mammalian somatic cells

  • Quite error prone

  • Essentially just ligates the 2 ends back together

• Homologous Recombination (HR)

  • More accurate

  • Uses sister chromatid as template

  • We process the blunt ends, such that the 5’ ends are resected (which is they are digested back a little), then the identical sister chromatid can be used as a template to repair this DSB (aka strand invasion)

What are the steps in Non-homologous end joining?

• Ku protein: a heterodimer that recognises and grasps the broken chromosome ends

• Protein complex forms, holding the two ends together

  • These proteins, including nucleases process the ends of the DNA (though not as much as HEJ)

• Then the Ku proteins on either end pull the two ends together

• DNA polymerase may be required to fill any gaps

• DNA ligase completes the process resulting in the end being covalently joined

• Results in a loss of nucleotides at the site of joining

  • This can lead to a deletion → mutations

What is Homologous recombination?

• Homologous Recombination involves the exchange of DNA strands between two homologous duplexes of DNA

• Duplexes: double helix DNA

• Homologous: identical or in this case, highly similar sequences

  • Sister chromatids (repair)

  • Homologous chromosomes (meiosis)

    • Very similar but have different alleles → still homologous

How is DNA base pairing critical for recombination?

• Homologous recombination only between DNA duplexes that have extensive regions of sequence similarity

• Two DNA duplexes engage in extensive base-pairing between a single strand from one DNA duplex and the complementary single strand from the other duplex

• A perfect match is not necessary but it must be close for homologous recombination to happen

Homologous recombination: functions

• Accurate repair of double stranded breaks: most widespread use of homologous recombination

• Genetic exchange between two homologous DNA sequences (DNA sequences similar or identical in nucleotide sequence)

• Mechanical role in assuring accurate chromosome segregation during meiosis in eukaryotes

Double stranded break repair via HR (homologous recombination)

• 1) Exonuclease chews/resects 5’ ends of the DS break

• 2) Strand exchange/invasion by homologous base pairing

  • One of the 3’ overhands will invade the intact sister chromatid/duplex → pairs with complementary bases → DNA synthesis using the 2nd duplex as a template

• 3) Extension of invading strand by DNA polymerase

• 4) Invading strand released, original helices reformed

  • Once released the extended region can then bind to the 2nd overhanging 3’ region (this is why it is important that those exonucleases at the start resect a large region either side of the break)

• 5) Gap filling by DNA polymerase

• 6) Ligation

*No loss of nucleotides

Double stranded break repair during replication

• 1) Nick or gap in parental DNA helix ahead of the replication fork

• 2) When fork reaches lesion, it falls apart: causing a DS break in one duplex, replication halts

• 3) Homologous recombination (strand invasion) restores DNA sequence using undamaged duplex as template

• 4) Replication can resume

How does a strand invade?

• Invading strand needs a way of disrupting a stable DNA helix of the other duplex (because the sister chromatid otherwise has no reason to open up), therefore we need protein it help this process

• The 3’ end of the invading strand is acted upon by RecA (E. coli) or Rad 51 (eukaryotes)

  • ATP bound RecA binds tightly to ssDNA forming a DNA-protein filament on the 3’ overhang that is going to invade → this actually stretches the DNA out a little bit exposing the nucleotides so that they can scan the duplex for complementary bases (in a sequence independent manner)

  • RecA also binds to the intact dsDNA helix and stretches (destabilise) it out, so that the ssDNA can invade

  • Once homologous sequence located (extended stretch of at least 15 nucleotides), strand invasion occurs

  • Single strand displaces one strand of the duplex and forms base pairs with the other strand resulting in a heteroduplex (pairing of two DNA strands from two different DNA molecules

  • Other proteins help in this process

• Destabilises the duplex DNA

What are Heteroduplexes?

• Strand invasion requires the pairing of a region of single-stranded DNA with a complementary strand in a different DNA double helix

• DNA hybridisation occurs between strands that are highly similar, but not necessarily identical

• Creates a region of DNA helix formed from strands that originate from two different DNA molecules – heteroduplex

• Some mismatch may occur

What can go wrong in Homologous Repair?

• Wrong template used (other chromosome homolog, not sister chromatid) lead to loss of heterozygosity

  • Mutations can become homozygous

    • if the mutant gene is used as a template, therefore we lose the functioning copy of our normal gene and we not are homozygous for a mutant

  • Can lead to cancer (e.g. tumour suppressor genes)

• Too little homologous recombination = increased mutation rate → can lead to cancer

  • Results from damage to portions in homologous recombination

  • Mutations in Brca1 and Brca2 cause breast cancer due to inefficient repair by homologous recombination

  • Brca2 helps to bring Rad51 protein to sites of damage and releases it in its active form onto ssDNA

Difference between mitosis and meiosis?

• dsDNA repair occurs shortly after DNA replication in the S and G2 cell cycle phases - when a daughter duplex can act as the repair template, (i.e. Before Mitosis)

• Meiosis differs from mitosis in that:

  • Two divisions,

    • Homologous chromosomes separate

    • Sister chromatids separate

  • Chromosomes in meiosis undergo recombination

    • Shuffles the genes

    • Different genetic combination in each gamete

  • Chromosomes in mitosis are identical

  • Outcome of meiosis is four (genetically unique) haploid cells

  • Outcome of mitosis is two (genetically identical) diploid cells

Steps in meiosis vs. mitosis

How can Recombination generate chromosome crossovers?

Recombination during meiosis

• Process differs slightly from Homologous Repair

• Begins with enzymes Spo11 and Mre11 creating a ‘double stranded break’ in one of the homologous chromosome

• Nucleotides cleaved/resected from the 5’ ends either side of the break

• Rad51 initiates strand invasion

• Second strand from nicked chromosome ‘captured’

• DNA polymerase and ligase synthesise new DNA and seal the gaps

• Results in a double Holliday junction structure

• Branch migration extends the cross over region or region of heteroduplicity

• Endonucleases cleave strands to resolve junctions

*Cross over is an exchange of genetic material between non-sister chromatids of homologous chromosomes

Key stages of Homologous Recombination: 1) Double stranded break either spontaneous or induced in one of the homologous duplexes

• In repair, this may begin as a nick, collapsing the replication fork

• During meiosis, enzymes Spo11 and Mre11 create a break

Key stages of Homologous Recombination: 2) 5’ ends of break processed leaving 3’ overhangs

Key stages of Homologous Recombination: 3) One of the two 3’ overhangs invades the undamaged duplex at the point where the sequence is complementary

• Rad51 in Eukaryotes

Key stages of Homologous Recombination: 4) DNA synthesised from the 3’ overhang, using the complementary strand as a template replacing the gap from the break

Key stages of Homologous Recombination: 5) Alternative pathways

• Invading strand release → what happens during repair

• Second Strand Capture → DNA recombination

Key stages of Homologous Recombination: 6) Invading strand release

• Broken double helix re-forms

• DNA polymerase fills any remaining gaps

• Ligation

• No crossover

• This mechanism used for repair

Key stages of Homologous Recombination: 6) Second Strand Capture

• Additional DNA synthesised to fill gap

• DNA ligated back together

  • 3’ end ligated back to the 5’ end of it’s original strand

  • BUT with a region now complementary paired to the homologous duplex

  • Double Holliday junction

    • Points where single strands separate from double helix: Branch Points

    • Points where strands crossover and join 4 DNA strands: Holliday junction

    • Branch points can move along the DNA strand and extend those regions of exchanged bases: branch migration

Second Strand Capture: Branch Migration

• An unpaired region of one of the single strands displaces a paired region of the other single strand, moving the branch point

• Increasing the regions of cross over/DNA exchange and heteroduplicity

• Specialised proteins and ATP hydrolysis ensure the branch moves in one direction

• This image only shows one branch point, IRL we have 2 branch points migrating in different directions

*Key stages of Homologous Recombination

Resolution of Holliday junctions

• Depending on how nucleases cut DNA, different product can arise, with or without crossover

• Crossover: exchange of chromatid segments

• Important to be able to identify crossover in the products

(image bottom)

• 2) The bottom strand has now rotated around

• 3a → vertical resolution results in a cross-over event

• 3b → horizontal resolution results in a non-cross over event

Recombination during meiosis

• Recombination definition: The natural formation in offspring of genetic combinations not present in parents, by the processes of crossing over of homologous sequences

• Repair-like event preferentially between maternal/paternal chromosome homologues

• Acts to ensure no two daughter cells are identical nor identical to parent

• Recombination can lead to gene conversion (bottom)

• Heteroduplexes resulting from mismatched bases following recombination

• Which is then detected by DNA repair systems

  • But these mechanisms cannot tell paternal/maternal apart

  • Therefore it will randomly choose a template strand and what strand will be corrected

  • Results in ‘conversion’ of one allele to another

  • Typically limited to small sections of DNA (part of genes)

Innate immune cells activate adaptive immunity

• B and T cells develop in the bone marrow and thymus respectively

• Naïve mature lymphocytes (B & T cells) constantly surveil the body for infection

  • Threats detected by B cell receptor (BCR) and T cell receptor (TCR)

• Importantly adaptive immunity has:

  • Immunological memory

  • Antigen specificity (focus here)

What are the common structural features shared by BCRs (antibodies) and TCRs?

• The antigen binding site is in the variable portion of the receptors

  • V = variable

  • C = constant

• The antigen binding site is contributed to by two different polypeptide chains

• The constant portion of the receptors confers functional attributes unrelated to antigen specificity

What are the differences in structural features between BCRs (antibodies) and TCRs?

• The BCR (membrane-bound antibody) consists of two heavy (H) and two light (L) chains linked by disulphide bonds

• The BCR can be secreted as an antibody by the B cell after it has been fully activated

• The TCR consists of one alpha (α) and one beta (β) chain

• The TCR is membrane bound and never secreted

BCR (antibodies) and TCR bind different antigenic determinants

• BCR → WHOLE antibody

• TCR → epitope is a peptide fragment buried within the antigen presented in the contex of MHC

• Antigen = molecule an adaptive immune response is directed against

• Epitope = part of antigen bound by a lymphocyte receptor (can have multiple epitopes)

Each lymphocyte receptor chain has three hypervariable regions

• The specificity of each BCR or TCR is unique

• The antigen binding site contains the most sequence variability

• These regions are known as hypervariable (HV) regions or complementarity determining regions (CDR)

• There are three HV regions on each chain (heavy, light, alpha, beta)

  • Therefore, for the antibody in the lecture we would have 12 HV regions

• The most variable of the three HV regions is HV3 in each chain

Where are Hypervariable regions located?

• The HV region isn't encoded by one gene. It's assembled by DNA recombination from smaller segments:

  • Heavy chain (and TCR β): V + D + J segments

  • Light chain (and TCR α): V + J segments only

• The hypervariable regions are found at the same point in each polypeptide chain

  • HV3 has the greatest variability of aa which means… (image below)

  • Amino acid sequence determines protein structure

  • Folding of loops determines antigen binding

• HV1 and HV2 are both encoded within the V gene segment itself. The V segment is long and contains the sequences for CDR1 and CDR2. So these two loops are determined by which V gene was picked. Diversity here comes from having many different V genes to choose from, but there's no extra junctional variation’

• HV3 sits right at the junction where the segments are stitched together. It includes the end of V, the whole D (heavy chain only), and the start of J. This is why it's the most diverse:

  • Many possible V, D and J combinations can be joined

  • During joining, nucleotides are randomly trimmed off and added at the junctions (junctional diversity, including N and P nucleotides)

So what do antigen receptors have to do with DNA recombination?

• We now know where lymphocyte receptor specificity is determined by hypervariable regions of antigen binding site

• But how is it generated?

• There are approximately 10¹² different B cells and 10¹² different T cells (i.e. lymphocytes with different receptors)

  • With each receptor being encoded by 2 chains (H/L or α/β) this would mean we need approximately 4×10¹² genes

  • But only 19,000–20,000 protein-coding genes in human genome! (not enough)

• So how does this work?

• Somatic DNA recombination!

The DNA in each of our somatic cells is the same?

• DNA recombination occurs during meiosis in the formation of ‘germ cells’ or gametes

• Recombination does not occur in our somatic cells (non-reproductive cells)

• DNA is preserved in an un-recombined form as the genetic blueprint for transcribing to mRNA and translation into required protein…..

• Thus each cell in your body has the same DNA sequence

• EXCEPT LYMPHOCYTES!!

Lymphocyte receptor diversity is generated through somatic recombination

• Lymphocytes are an exception to the “no recombination” rule (in mitosis)

• DNA recombination (somatic recombination) does occur in lymphocytes

• Involves physically cutting out small regions of DNA and recombining these to create unique truncated sequences

  • Your genome doesn't contain a ready-made gene for every possible antibody. It contains a library of short gene segments, labelled V, D and J, and each B or T cell picks one of each and stitches them together. The hypervariable regions (HV1, HV2, HV3, the loops that touch the antigen) are built from pieces of these segments

• The unique DNA sequences encode unique polypeptide chains that create unique lymphocyte receptors

• So which sequences are being recombined?

Lymphocyte receptor loci composed of multiple gene segments

• Lymphocyte receptor chain loci composed of multiple, distinct, V(D)J gene segments

  • V = variable

  • D = diversity (look how the light and alpha chains doesn’t have this)

  • J = joining

  • C = constant

• Similar germline sequences in all loci

Receptor loci vary in somatic cells versus mature lymphocytes

• We can see that only one section from each region has been selected in the mature B cell and have been combined into a unique receptor

DNA sequence differs in a B cell clone versus a somatic cell

• Somatic cells each carry the DNA ‘blueprint’ inherited from the parents

• DNA in each mature B cell clone has been rearranged (“recombined”) with loss of a large amount of the genetic information

• Same recombination process occurs in B and T cells

• Lymphocyte receptor DNA sequence differs between clones

• All progenitor lymphocytes share the same ‘blueprint’ DNA

• In each B or T cell clone V(D)J gene segments are selected at random

• Therefore each clone has a different rearrangement of their lymphocyte receptor locus

Steps in lymphocyte recombination

• BCR heavy chain gene locus in germline DNA

• Recombination first occurs between Diversity (D) and Joining (J) regions

  • The first recombination event joins one D segment to one J segment. The DNA between them is cut out and discarded, along with the other D and J segments in that stretch. The cell now has a DJ unit.

• Recombination next occurs between Variable (V) and DJ regions

  • One V segment is joined to the DJ unit, again deleting the DNA in between. Now the cell has a complete VDJ exon

• The recombined gene is transcribed to RNA

  • At this point the RNA contains the VDJ unit, then any leftover J segments that weren't used, then the C region sequences

• The VDJ complex is spliced onto the Constant (C) region RNA

  • Extra J regions are spliced out

• mRNA is translated to produce the BCR heavy chain polypeptide

*Light chain is also being transcribed/translated at the same time just without the D segments

umber of possible receptors depends upon number of combinations of V(D)J gene segments possible

• Immunoglobin → BCR (Heavy chain + 2 diff types of light chains)

So how does recombination relate to antigen binding site specificity?

• Hypervariable regions are encoded by the Variable (V), Diversity (D), and Joining (J) gene segments

• Hypervariable regions 1 and 2 are encoded within V gene segments

• Hypervariable region 3 is encoded by a combination of V, D and J for

  • BCR heavy chain

  • TCR beta chain

• Hypervariable region 3 is encoded by a combination of V and J for

  • BCR light chains

  • TCR alpha chain

• Each cell randomly picks different V, D and J segments, and since HV1, HV2 and HV3 are built from them, each cell ends up with a different set of loops

• The loops together form the antigen-binding site, so a different shape means a different antigen recognized

• Thus somatic recombination impacts protein function (i.e. antigen binding site specificity)

What mechanisms contribute to Lymphocyte receptor diversity?

• 1) Recombination of lymphocyte receptor gene segments (VDJ)

• 2) Nucleotide deletion at junctions

• 3) Addition of nucleotides at junctions

Somatic recombination occurs during B and T cell development

• B cells rearrange their BCR in the bone marrow

• T cells rearrange their TCR in the thymus

• This process occurs before the lymphocytes have encountered the antigen that their receptor binds to

• Recombination of the BCR and TCR genes involves coordinated activity of several enzymes

• Recombination activating gene (RAG) 1 and 2 enzymes

  • Expressed only in developing B and T cells

• DNA repair enzymes

  • Found in all cells and involved in DNA damage repair

The mechanism of V(D)J rearrangement

• RAG enzymes recognise specific Recombination Signal Sequences (RSS) in lymphocyte receptor genes to initiate recombination

• RSS are found…

  • 3' of each V gene segment

  • 5' of each J gene segment

  • Both sides of each D gene segment

• RSS sites consist of a…

  • Conserved 7 nucleotide heptamer

  • 12 or 23 non-conserved nucleotide spacer

  • Conserved 9 nucleotide nonamer (to allow enzyme binding)

Recombination only occurs between RSS sites that have 12 bp and 23 bp spacers

• Using BCR heavy chain as an example:

• A D_H gene segment can be joined to a J_H gene segment

• A V_H gene segment to a D_H gene segment

• V_H gene segments CANNOT be joined to J_H gene segments directly (heavy chain)

• As both V_H and J_H gene segments are flanked by 23 bp spacers

• 12/23 rule (opposites 12 OR 23 attract - 23 and 23 will not attract/bind)

• Recombination only occurs between segments flanked by 12 bp spacer and 23 bp spacer

• Ensures that recombination will not occur between only V elements (or only J elements)

Rag 1 and Rag 2 enzymes mediate the synapse of two RSS sites with 12 bp and 23 bp spacers and cleave

• Recognition of RSS sites by RAG1:2 recombinase enzymes

• Binding of RAG enzymes and bringing V(D)J gene elements together

• Cleavage of DNA fragments

• Binding accessory proteins Ku70:Ku80 to cleaved ends

• DNA ligase IV and XRCC4 bind to Ku accessory proteins (ends of the gene segments) to form the coding joint

• Ligation of DNA ends to form the new V(D)J segment (coding joint)

  • Excess DNA in signal joint lost upon subsequent cell division

Junctional diversity introduces further diversity into HV3

• 1) Recombination of lymphocyte receptor gene segments (VDJ)

• 2) Nucleotide deletion at junctions

• 3) Addition of nucleotides at junctions

• Combinatorial diversity can generate ~1.6 × 10⁶ different BCRs

  • Combinatorial diversity comes from which segments get picked. You choose one V, one D and one J from the library, and different choices give different antibodies

  • HV1 and HV2 sit inside the V segment, away from the joints, so they get combinatorial diversity only

    
    

• Junctional diversity can increase this by a factor of ~3 x 10⁷

  • Junctional diversity comes from how the segments are joined. At each joint (D to J, then V to DJ), the cell randomly alters the DNA ends before sealing them. So even if two B cells pick the exact same V, D and J, their junctions can still differ

  • Introduces diversity into HV3

• Combined, the number of unique lymphocyte receptors approaches ~4.8 × 10¹³ (48 trillion)

• Changing the number of bases can also shift the reading frame. Roughly two thirds of rearrangements end up out of frame and non-functional, so the cell has to try again or die. This is the cost of the system

Junctional diversity is created by the addition and subtraction of nucleotides to the coding joint

• RAG1/2 binds RSS creates DNA double stranded breaks

• Artemis: DNA phosphokinase (DNA-PK) opens hairpins to form random overhangs

  • Palindromic or P-nucleotides

• Terminal deoxynucleotidyl transferase (TdT) adds random nucleotides

  • Non-templated or N-nucleotides

• Exonuclease removes random nucleotides

  • The removing and addition of nucleotides introduced variability

  • They also change reading frame - encoding novel amino acids

• (bottom) Pairing of overhanging strands, DNA ligase IV and XRCC4 ligate strands back together

• Exonuclease removes unpaired nucleotides

• DNA polymerase “fills in” overhangs

• New “DJ” segment!

  • Not always functional

  • Introduce frame shifts with stop codons → thus producing non-functional/truncated proteins

  • Encode molecules with different amino acid composition and thus structure