DNA Replication Flashcards
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.