DNA replication, repair & gene expression Flashcards
What is the flow of genetic information (2)
- Replication → DNA → RNA via transcription → Protein via translation
- Information must not only be transmitted from one generation to another but be expressed with high fidelity (accuracy) and speed
What is the cell cycle (5)
- G0 - cells not dividing
- G1
- S - DNA replication, 2 copies of the genome
- G2 - call prepares for division
- M - 2 duplicate daughter cells
What are phosphodiester bonds (3)
- Link two nucleosides
- make up the negatively charged, hydrophilic phosphate-(deoxy)ribose backbone
- on the 5’ carbon of one sugar and the 3’ carbon of another.
What is DNA replication (2)
- SEMICONSERVATIVE
- BIDIRECTIONAL - always in 5’ → 3’ direction
What does DNA replication require (5)
- DNA polymerase
- Template DNA
- Deoxyribonucleoside 5’-triphosphate Proofreading enzyme
- Primer – short RNA sequence
- Protein factors and enzymes
How does cell replication occur at different rates (3)
- Some cells constantly divide
(hair, fingernails and bone marrow cells) - Other cells go through several rounds of cell division and stop (specialised cells including brain, muscle and heart)
- Some cells stop dividing but can be induced to divide to repair injury (skin cells and liver cells)
What mistakes in replication can occur (4)
- Substitutions
- Misincorporation
- Tautomers - mismatching
- Slippage - repetitive DNA, secondary structures
What spontaneous damage may there be to DNA (2)
- Bass loss - backbone still intact, mutations, purines more sensitive
- Deamination - convert C-U instead of G, etc.., results in mutation
What causes spontaneous damage to DNA (3)
- Spontaneous or enzymatic conversions
- Free radical species (oxidative stress)
- Reactive oxygen species (ROS) damage bases
What causes environmental DNA damage (3)
- Radiation - X-rays, gamma-rays
- Non-ionising - UV light
- Carcinogens
How does radiation cause environmental DNA damage (3)
- Secondary damage ROS generated
- Damages bases
- Double strand breaks
How does non-ionising UV light cause environmental DNA damage (3)
- Bases absorb energy within the UV range
- Photoactivates bonds - covalent bonds between adjacent bases
- Distorts DNA blocking transcription & replication leading to mutation
How do carcinogens cause environmental DNA damage (3)
- Cause DNA adducts - block transcription & replication leading to mutation during replication
- Result in intra/inter-strand crosslinks
- Sometimes product of cellular metabolism
What are the mechanisms to repair DNA damage (7)
- Direct reversal
- Single-strand break repair
- Base excision repair
- Nucleoside excision repair
- Mismatch repair
- Double-strand break repair (non-homologous)
- Homologous recombination
What is direct reversal DNA repair (2)
- Most efficient way to deal with damage
- not most common though
What is single-strand break DNA repair (2)
- most common
- the cellular responses and repair of SSB are not well understood
What is base excision DNA repair (2)
- Damage is recognised and the base is removed by DNA glycosylase enzymes (different one for each type of damage)
- DNA polymerase and DNA ligase enzymes add the correct base
What is nucleotide excision DNA repair (2)
- Recognises distortion in DNA (more
flexible than BER) - Multiple proteins involved
What is mismatch DNA repair
Unlike NER/BER, not obvious which strand of DNA is damaged, and which should be used as template
What is double-strand break DNA repair (4)
- Non-homologous end joining
- Less accurate, but the primary pathway in vertebrates (is more efficient)
- If defective can lead to genetic disorders (e.g. severe combined immunodeficiency SCID)
- Damage in G1 phase of cell cycle
What is homologous recombination DNA repair (3)
- Needs a long homologous sequence as a template (sequences are exchanged)
- Result in ‘new combinations of DNA’ – genetic variation (e.g. BRCA1, BRCA2 gene)
- damage in the S phase in the cell cycle
What is gene expression (3)
- expressing gene information into gene products (e.g. RNA molecules)
- Transcription: DNA → RNA
- Translation: mRNA → protein
What is transcription (8)
- Only particular gene(s) are transcribed at any time, depending on the requirements of the cell
- Occurs in the nucleus (eukaryotic cells)
- Makes use of COMPLEMENTARY base pairing
- RNA-DNA hybrid double helix temporarily formed
- No proof-reading of the RNA strand, less detrimental if errors occur
- High levels of regulation of transcription are present – to avoid unnecessary energy waste
- Post-transcriptional modification of RNA (splicing, 5’RNA-capping, 3’ polyadenylation)
- RNAs transported from the nucleus to the cytoplasm
What does transcription require (5)
- RNA polymerases
- DNA template
- Ribonucleoside 5’-triphosphate
- Transcription factors & enzymes
- Energy
What are the types of RNA polymerases (3)
i - makes ribosomal RNA
ii - makes messenger RNA
iii -makes transfer RNA
How is transcription regulated
Cells have tight control over which genes get expressed at any one time
What is translation (10)
- Occurs in the cytoplasm (rough endoplasmic reticulum) on ribosomes
- 4 letter nucleic acid language → 20 letter protein language
- tRNA translates the 3 base codon into amino acid sequence
- Aminoacyl-tRNA synthetases ‘charge’ the tRNA with the corresponding amino acid
- Ribosomes associate around the mRNA
- tRNAMet occupies the P-site
- Appropriate aminoacyl-tRNA base pairs with the codon at the A-site
- Peptide bonds are formed between two amino acids
- Ribosome moves by one codon, releasing an ‘empty’ tRNA
- Elongation of the peptide chain continues…
- Polysomes – several ribosomes can translate mRNA at the same time.
- Most genetic information pathways finish with proteins (note rRNA, tRNA are final products).
- Proteins undergo post-translational modification before they are transported to their functional destination.
What does translation involve (4)
- mRNA
- Ribosomes (proteins + rRNA)
- Charged tRNA= tRNA + amino acids
- Energy
What are DNA replication inhibitors used as (2)
- Anticancer
- Antiviral
What drugs affect gene expression (2)
- Target transcription factors TF (in cancer therapy)
- RNA interference (gene silencing by dsRNA)
How do target transcription factors work
inhibit TF synthesis
How fo RNA interference drugs work (2)
- Synthetic small interfering RNA (siRNA) (e.g. bevasiranib)
- Targeting age-related macular degeneration, AMD
How do drugs that affect gene expression work (3)
- Modulate the physiological responses
- increasing or decreasing the transcription of key genes (production of ‘disease-causing’ proteins).
- Still in clinical trials
What are sources of DNA damage (3)
- Environmental (Radiation, carcinogens)
- Mistake in replication (Substitution, slippage)
- Spontaneous damage (free radical, reactive oxygen species)
