DNA replication, repair & gene expression

Question 1

What are the main processes occurring during DNA replication (8)

Answer 1

• Helicase splits DNA cells
• DNA polymerase replicates DNA molecules to build new strands of DNA in the 5’ to 3’ direction of the new strand.
• Primase signals where polymerase should start replicating
• Ligase glues DNA fragments together
• Is SEMICONSERVATIVE - since each strand has an original strand and a new strand
• Is BIDIRECTIONAL, always in 5’ → 3’ direction
• Different types of cells replicated their DNA at different rates
• On the lagging strand each fraction where primase has to start is called Okazaki fragments

Question 2

What are the types of DNA damage (7)

Answer 2

• Radiation – Ionising: X‐rays, gamma‐rays (medical sources). Damages bases (e.g. 8‐oxo-dG). Strand breaks are often clustered, thus a source of double-strand breaks
• Non-ionising: UV light - Bases absorb energy within the UV range. Photoactivated base resulting in covalent bonds between adjacent bases distorting DNA, which can block transcription replication, leading to mutation
• Carcinogens – Causes DNA adducts (section of DNA bound to a cancer-causing chemical). Can result in intra- and inter-strand crosslinks: Alkylating agents (reactive carbon-containing chemicals). Not always due to direct exposure: sometimes carcinogen is a toxic product of cellular metabolism. Example: cigarette smoke the breakdown products (benzo(a)pyrene-7,8-diol-9,10-epoxide-N2-deoxyguanosine). Adducts are bulky and block transcription and replication; they can interfere with base pairing and introduce mutation during replication
• Substitutions - can cause mismatching
• Slippage - causes repetitive DNA, secondary structures
• Deamination – Altered base has a different base (Converts C to U, etc.)
• Base loss – Causes mutation, can lead to strand breaks (single or rarely double-strand breaks)

Question 3

What are the mechanisms involved in DNA repair (7)

Answer 3

• Direct reversal –Most efficient way to deal with damage – not most common though
• Single-strand break (SSB) repair (SSBR) –most common, but the cellular responses and repair of SSB are not well understood
• Base excision repair (BER) – Damage is recognised and base removed by DNA glycosylase enzymes (different one for each type of damage). DNA polymerase and DNA ligase enzymes add correct base
• Nucleotide excision repair (NER) – Recognises distortion in DNA (more flexible than BER) with multiple proteins involved
• Mismatch repair (MMR) – Unlike NER/BER, not obvious which strand of DNA is damaged, and which should be used as template
• Double-strand break (DSB) repair (DSBR) - Non-homologous End joining (“NHEJ”). Less accurate, but the primary pathway in vertebrates (more efficient). If defective can lead to genetic disorders (e.g. severe combined immunodeficiency SCID)
• Homologous recombination (“HR”) - Needs a long homologous sequence as template (sequences are exchanged). Results in ‘new combinations of DNA’ – genetic variation (e.g. BRCA1, BRCA2 gene) Which DSBR mechanisms used depends on stage in cell cycle (NHEJ – in G1 phase; HR – in S phase)

Question 4

What are the main processes occurring during DNA transcription (7)

Answer 4

1. Double helix is broken apart by DNA helicases
2. RNA primase, acts as a primer for replication to start
3. RNA polymerase uses COMPLEMENTARY base pairing to produce RNA from 3’ to 5’ so RNA is synthesised from 5’ to 3’
4. Ligase sticks together the Okazaki fragments

Eukaryotic cells have three types of RNA polymerases (each make a different type of RNA):

1. RNA polymerase I – makes Ribosomal RNA (rRNA) – structural component of the ribosomes (protein synthesis ‘machinery’)
2. RNA polymerase II – makes Messenger RNA (mRNA) – carries the sequence that is encoded by the genes to be expressed
3. RNA polymerase III – makes Transfer RNA (tRNA) – adapter that reads the mRNA sequence and transfers the appropriate amino acid

Question 5

What are the main processes occurring during RNA translation (7)

Answer 5

1. Ribosomes associate around the mRNA in the cytoplasm (rough endoplasmic reticulum) on ribosomes
2. tRNAMet occupies the P-site
3. Appropriate aminoacyl-tRNA base-pairs with the codon at the A-site ‘charging’ the tRNA with the corresponding amino acid
4. Peptide bonds are formed between two amino acids
5. Ribosome moves by one codon, releasing an ‘empty’ tRNA
6. Elongation of the peptide chain continues…
7. Proteins undergo post-translational modification before they are transported to their functional destination.