DNA replication Flashcards
What is RNA polymerase used for?
Transcription
What is DNA polymerase used for?
DNA replication
How are DNA polymerases isolated?
Use biochemical methods to split into fractions. Test fractions with TTP incorporation assay to find DNA polymerase.
How do DNA polymerases work?
Product template directed, with a very low error rate (1 in 10^9 nucleotides copied). dNTPs are activated precursors for DNA synthesis with high energy phosphate bonds. Synthesise in 5’ to 3’ direction.
Requirements for DNA replication?
Single stranded DNA template. DNA helicase (requires ATP) DNA primase. A single stranded binding protein to protect the ssDNA. Telomerase binds to the ends of chromosomes and adds short repeats copied from internal RNA molecule - needed due to the end replication problem with primers on the lagging strand. dNTP building blocks. DNA/RNA primer as DNA polymerase can’t start de novo.
What is the DNA replication fork?
The Y shape formed at the replication origin. As DNA is only synthesised in the 5’ to 3’ direction, there are fragments of DNA found on the lagging strand which need to be joined together with DNA ligase. Needs a primer enzyme to continually add primers in.
How is the high fidelity of chromosome replication achieved?
DNA polymerases are also involved in DNA repair, they fill in the gaps left by damage to DNA after the cell has removed a damaged nucleotide.
No proofreading in repair as there is less chance of a mistake.
DNA polymerase has a proofreading ability, use an intrinsic 3’ to 5’ exonuclease activity to remove acidentally added nucleotides. When an incorrect base pair is recognized, DNA polymerase reverses its direction by one base pair of DNA and excises the mismatched base. Following base excision, the polymerase can re-insert the correct base and replication can continue.
What is an exonuclease?
Exonucleases are enzymes that work by cleaving nucleotides one at a time from the end (exo) of a polynucleotide chain.
A hydrolyzing reaction that breaks phosphodiester bonds at either the 3’ or the 5’ end occurs.
Differences between eukaryotic and prokaryotic replication?
