When in the cell cycle does DNA replication occur and how long does it last?
S phase
8 hrs
What are replication forks?
Y shaped structures where new DNA strands are synthesised by the replicosome (multi-enzyme complex).
Where does DNA replication begin?
At origins of replication - 1 in prokaryotes, multiple in eukaryotic chromosomes due to greater size of genome.
Describe the steps in DNA replication?
- Helicases unwind the parental double helix.
- Single-strand binding proteins stabilise the unwound parental DNA.
- The leading strand is synthesised continuously in the 5’-3’ direction by DNA polymerase.
- The lagging strand is synthesised discontinuously: primase synthesises a short RNA primer which is extended by DNA polymerase to form an Okazaki fragment.
- After the RNA primer is replaced by DNA (by another DNA polymerase), DNA ligase joins the Okazaki fragment to the growing strand.
What is the DNA replication reaction driven by?
Pyrophosphate hydrolysis (to add a dNTP to the growing DNA strand)
What are DNA polymerases?
Enzymes that create DNA molecules by assembling nucleotides in a complementary way to a template DNA strand.
In which direction does DPol synthesise DNA and read the template DNA?
- Reads template strand in 3’-5’ direction.
- Assembles new DNA strand in 5’-3’ direction.
What does DNA polymerisation require to begin?
A pre-existing RNA primer from which to extend.
How does DPol reduce its replication error rate?
- Contains an exonuclease domain: detects base pair mismatches and removes the incorrect nucleotide (to be replaced by the correct one).
- Reduces error rate from 1/10^6 to 1/10^9.
What can be the result of mutations in the DPol exonuclease domain?
- Tumours
- E.g. Ultramutated group of endometrial carcinomas characterised by mutations in DPolE exonuclease domain.
What are endonucleases and exonucleases?
Both are nuclease enzymes that catalyse the hydrolysis of single nucleotides present in a DNA chain at specific sequences:
- exonucleases cleave nucleotides at the ends of the DNA molecule (requires free 3’ or 5’ hydroxyl group)
- endonucleases cleave bonds within the molecule
What is DNA replication stress?
Inefficient replication that leads to fork slowing, stalling and/or breakage.
What are the consequences of errors in DNA replication?
Can lead to genomic instability - can cause either mutations or cell death depending on context - both can cause disease.
Describe the factors causing DNA replication stress.
- Defect in replication machinery (e.g. Helicase stalling)
- Factors hindering replication fork progress, e.g.
- DNA lesions
- DNA secondary structure
- Repetitive DNA
- Fragile sites or oncogene-induced stress
- Defects in the response pathways to the above factors, e.g.
- DNA repair pathways
- Regulation of origin firing
How does splippage of the replication machinery cause errors?
- Slippages occur at trinucleotide repeats - microsatellites - due to repetitive nature of sequence.
- 2 scenarios:
- newly synthesised strand loops out - 1 nucleotide is added on the new strand - extra nucleotide.
- template strand loops out - 1 nucleotide is omitted on the new strand - missing nucleotide.
What is the result of defects in the exonuclease activity (3’-5’) of DPol?
Increased mutation rate as mismatches aren’t removed.
Which defects allow the persistence of DNA strand breaks?
- Base excision repair (BER) defect causes DNA single-strand breaks to persist.
- BRCA deficiency causes DNA double-strand breaks to persist, e.g. In some forms of breast and ovarian cancer.
Why are DSBs the most ‘dangerous’ types of DNA damage?
- Disrupt molecular continuity
- Difficult to resolve
- Can lead to mutations or cell death if not repaired correctly. Mutations include translocations and chromothrypsis (chromosomal rearrangements)
Which hypothesis explains why age is the biggest risk factor in cancer?
Mutation accumulation hypothesis
Which disease is associated with a mutation in helicase?
- Werner protein is a helicase important during DNA replication; defects lead to DNA replication faults and DNA damage.
- WERNER SYNDROME involves autosomal recessive WRN gene mutations.
- Symptoms: premature display of features associated with normal ageing, inc. cataracts, skin ulcers, type 2 diabetes, atherosclerosis, osteoporosis and cancer predisposition.
Suggests endogenous and exogenous sources of DNA damage.
Endogenous:
- DNA replication errors
- V(D)J recombination
- Free radicals
Exogenous:
- Ionising radiation
- Alkylating agents
- UV light
- Anti-cancer agents
What is the rate of DNA damage from environment and normal metabolism?
Up to 1 x 10^6 molecular lesions/cell/day.
Suggest examples of DNA damage and their causes.
- Apurinic/apyrimidinic site: spontaneous DNA loss due to label nature of N-glycosidic bond linking nucleobase and pentose sugar.
- Deamination (loss of amine group).
- Mismatches: replication error.
- Single strand breaks
- Double stand breaks: free radicals generated as a byproduct of metabolism or exposure to exogenous physical agents (e.g. Ionising radiation)
- Pyrimidine dimer: non-ionising UV radiation
- Bulky adducts or inter- & intra-strand crosslinks: chemical compounds (e.g. Cisplatin): block DNA replication
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Enzyme Activity19
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Protein Function15
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DNA Replication23
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Mitosis & Meiosis12
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DNA Repair23
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DNA & RNA17
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Genes & The Genome8
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Mutations22
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Spontanous Mutations - Early Gametogenesis16
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Molecular Techniques - DNA27
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Inheritance13
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Molecular Techniques - Chromosomes14
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Molecular Techniques - Proteins10
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Protein Primary Structure18
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Protein Structure 213
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Protein Bonds In Protein Structure12
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The Clotting Cascade20
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Basic Cell Structure29
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Post-translational Protein Processing13
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Protein Targeting12
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Chromosomal Abnormalities25
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Gene Expression20
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Enzymes (new)0
