Point mutation:

Refers to changes in the sequence of 1 base pair in a DNA sequence; includes missense (aa substitution), non-sense
mutations (subs a stop codon for aa), substitution of 1 or few bases. Ex: in sickle cell anaemia, single nucleotide
change from T to A -> aa change from Glu to Val -> affects RBC shape to sickle -> oxygen carrying capacity decreases.
Crohn’s disease, an insertion of C nucleotide at position 3020 leads to premature stop codon, shortening the protein
to be transcribed. Causes inflammation of digestive tract.

Frameshift mutation:

Occurs when there’s any insertion/deletion of 1 or more nucleotides in a DNA sequence not divisible by 3. This
changes the reading frame of the base sequence. Insertions and deletions remove and add nucleotides, respectively,
resulting change in protein structure and function. Causes more significant effects than point mutations. Ex: Cystic
fibrosis, Two frameshift mutations (one is the insertion of two nucleotides and the other deletion of one nucleotide)
in the CFTR genes result in cystic fibrosis.

Silent mutation:

Change of a single nucleotide doesn’t affect and change the aa and therefore the protein bcos the mutated codon
codes for the same aa.

Repeat expansion:

Nucleotide repeats are short DNA sequences that are repeated a number of times in a row. A repeat expansion is a
mutation that increases the number of times that the short DNA sequence is repeated. This type of mutation can
cause the resulting protein to malfunction. Ex: Huntington disease. More than 36 repetitions of CAG sequence

Base excision repair:

1. Used to correct damaged DNA bases

2. Repairs this damaged DNA bases caused by oxdn, alkylation, or hydrolysis
3. Damaged bases removed by DNA glycosylase -> AP (apurinic/apyrimidinic) site formed in DNA
‒ AP site represent damaged section of DNA
4. AP site recognized by AP endonuclease -> creates a nick in damaged section of DNA -> AP sites cleaved off -
> yielding exposed 3’OH
5. DNA pol. inserts the missing bases by complementary base pairing (A-T, G-C)
6. DNA ligase seals the nick -> dsDNA repaired :)

Nucleotide excision repair:

1. Used to remove whole nucleotide seq.

2. Repairs DNA caused by exogenous mutagens (UV light)
3. Pyrimidine dimer (T=T) forms as result of UV light exposure -> forms a bulge in the DNA
4. Bulge cleaved off by excision nuclease & a length of 10-20 seq of nucleotides also cleaved along with the
bulge
5. DNA helicases bind to cleavage site -> unwinds -> removes cleaved DNA segment (incl. the 10-20
nucletoides) -> big gap on the DNA strand
6. To fill the gap, DNA pol. binds to cleavage site and does step 5 and 6 from prev mechanism
DNA mismatch repair:

1. MutS detects mismatch -> combine with ATP molecule -> activated -> forms ATP-MutS complex
2. This complex recruits MutL (carrier protein complex)
3. MutS + MutL = MutS-MutL complex
‒ MutS-MutL complex bend the DNA -> activate MutH
‒ Forms MutS-MutL-MutH complex
4. MutH of this complex (MutS-MutL-MutH) cleave off DNA segment having the mismatch
5. DNA helicases bind to cleavage site -> unwinds -> removes cleaved DNA segment -> big gap on the DNA
strand
6. To fill the gap, DNA pol. binds to cleavage site and does step 5 and 6 from prev mechanism

dsDNA strand break repair:

1. Occur as a consequence of ionising radiation

2. Repair by homologous recombination (HR) and RAD proteins involved
3. HR begins with the MRE11/RAD50/NBN (MRN) complex binding to the broken ends of the double-stranded
break (DSB)
4. MRE11 excises nucleotides from the 5′ ends leaving 3′ ends stranded at the DSB break points.
5. The 3′ ends coated w/ replication protein A (RPA)
6. RPA replaced by RAD51 in a reaction promoted by mediator proteins, such as BRCA2
7. Rad51-DNA-nucleoprotein filament produced -> this filament invades at the homologous site on ds
homologous sister chromatid
8. Holliday junction develops at this site -> the homologous sister chromatid used as a template to perform
DNA repair (same as step 5 and 6 from prev mechanisms)
9. Holliday junction resolved to form crossover and non-crossover products -> DNA repair is complete

Insulin receptor (IR) structure and signalling pathway:

Structure:

- RTK family; 2 extracellular alpha-subunits linked to 2 beta-subunits and to each other by disulphide bonds.
- A-subunit: cysteine-rich domain; b-subunit: extracellular transmembrane and cytosolic domain
- IR binds to phosphorylated residues on large docking proteins known as the insulin receptor substrate family
(IRS1-6) and also adapter protein Shc (Src homology 2 domain containing)

Pathways:

Insulin bind to the extracellular α subunits of the IR -> induce conformational change -> results in the
autophosphorylation of tyrosine residues in the β subunits -> these form the binding sites for IRS proteins

2 pathways result from the insulin receptor-IRS interaction: Ras/MAPK (Ras dependent) and PI3K/AKT (Ras
independent)

Ras/MAPK:

1. binding of Grb2 to tyrosine-phosphorylated Shc, or through Sh2 binding to the insulin receptor.
2. Grb2 binds to proline-rich regions of proteins such as son-of-sevenless (SOS)
3. SOS Ras-GDP to Ras-GTP via phosphorylation.
4. Activated Ras-GTP stimulate downstream effectors like Raf kinases
5. Raf kinase activate its downstream targets MEK1 and MEK2
6. MEK1 and 2 phosphorylate and activate the MAP kinases, ERK1 and 2.
7. Activated ERK1/2 are directly involved in cell proliferation and differentiation.

PI3K/AKT:

1. Activated when PI3K regulatory subunits bind to IRS1 and IRS2 -> PI3K catalytic subunit, p110, activated.
 2. PI3K catalyzes the phosphorylation of PIP2 to produce PIP3 at the cell membrane.
3. PIP3 act as second messenger and recruit PDK1 and AKT to the membrane
4. PDK1 phosphorylation activates AKT residues.
5. AKT plays a role in four critical downstream processes
a) regulation of protein synthesis via the substrate protein mTOR
b) regulation of glycogen synthesis GSK3. GSK3 inhibited when phosphorylated by AKT, results in glycogen
synthesis.
c) regulation of gluconeogenic and adipogenic genes through the transcription factor FOXO1. In absence of
insulin, FOXO1 translocates to the nucleus -> activates gene expression involved in gluconeogenesis.
d) regulates translocation of the insulin-sensitive glucose transporter GLUT4, in intracellular vesicles of
muscle cells and adipocytes to the cell membrane via exocytosis. GLUT4 facilitates glucose uptake from
the blood into cells.

ROS and cell apoptosis

Mention only if asked:

A free radical: molecular species existing independently having one or more unpaired electrons in its outer orbit.
Presence of an unpaired electron in the outermost shell of a free radial, the odd number of electrons in it make the
species highly reactive, unstable and have a shorter half-life. They can remove electrons from other atoms or
molecules to achieve stability due to their high reactivity. As a result, the atom or molecule being attacked loses one
of its electrons and becomes a free radical itself, thereby triggering a chain reaction cascade that eventually damages
the living cell

- The ROS has dual purpose in the biological system, acting as both helpful and harmful substances in the
body.
- ROS positive effects at low or moderate levels and involved in defence against pathogenic microorganisms,
numerous cellular signalling pathways, inducing mitosis in cells (mitogenic response), and regulation of
redox reactions.
- ROS cause oxidative stress at higher concentrations damaging biomolecules.
- ROS: hydroxyl radical (OH•), superoxide anion radical (O2•‒), hydrogen peroxide (H2O2), oxygen singlet
(1O2), hypochlorite

- The hydroxyl free radical can be generated in two reactions: Fenton reaction, or Haber-Weiss reaction. In the
Fenton reaction H2O2 reacts with metal ions (Fe2+) and is generally bound in protein complexes like ferritin.
Haber-Weiss reaction, HO• is generated by the reaction of O2•‒ and H2O2.

Lipid peroxidation: free radical attacks and removes H from methylene groups in a fatty acid -> lipid radical
formation.

Lipid radical reacts with O2 molecule = lipid peroxyl radical; lipid peroxyl radical removes a hydrogen atom from
another lipid molecule producing a new L• continuing the chain reaction.

The resultant lipid peroxyl radical undergoes re-arrangement generating endoperoxides which eventually form toxic
end products of lipid peroxidation that are able to cause DNA and protein damage such as malondialdehyde (MDA)
and 4-hydroxynonenal (4-HNA).

DNA damage: ROS, particularly the HO•, directly interacts with all DNA components, including purine and pyrimidine
bases and the deoxyribose sugar backbone, causing modifications such as single and double stranded breaks in the
DNA. Induces p53 tumour suppressor protein-> cell apoptosis
Protein damage: Sulphur-containing amino acids like methionine and cysteine are more prone to oxidation by
oxidative free radicals transforming them to disulphides and methionine sulphoxide. Carbonyl residues are formed.
Secondary damage caused by toxic by products of lipid peroxidation.

Apoptosis:

‒ MAPKs such as ERK, p38, and JNK serve a central role in ROS-mediated apoptotic signalling.
‒ p53 regulate apoptosis by down-regulating proteins such as Bcl-2. p53 activates the transcription of pro-
apoptotic genes that are crucial for inducing the intrinsic pathway of apoptosis, such as Bax, Bid, and Apaf-1,
also extrinsic pro-apoptotic factors such as Fas and FasL.
‒ cytosolic p53 translocate to mitochondria and interact directly with pro-apoptotic proteins Bax and Bak,
allowing mitochondrial outer membrane permeabilization (MOMP), release of pro-apoptotic factors and
apoptosis followed by caspase initiator (Cas9) -> protein substrate cleavage -> Pro-caspase 3 -> executioner
capsase activation (Cas3) -> apoptosis.

Free radicals counterattacked by antioxidants.

1. free radicals react rapidly with oxygen to produce superoxide -> undergoes dismutation to generate H2O2,
catalysed by Superoxide dismutase SOD
2. H2O2 detoxified by antioxidants such as catalase and glutathione (GSH) (cofactor of GP)/glutathione
peroxidase
3. GSH acts as antioxd. in nucleus, mitochondria and cytosol
4. GSH: a) Participates in amino acid transport through plasma membrane; b) Regenerate the antioxidants
vitamins C and E back to active form.
5. Vit C reduces lipid peroxidation. Vit E places itself into cell membranes and protect unsaturated fatty acids