Introduction, Evolution and Genomes-9 Flashcards
Enzymology of DNA replication
What does the Watson-Crick base pairing model underly?
DNA replication is semi-conservative.
DNA strands are anti-parallel.
What direction is DNA synthesised?
New DNA is synthesised in the 5’ -> 3’ direction.
Describe DNA synthesis
DNA synthesis is semi-continuous, with a leading strand and a lagging strand.
The leading strand is replicated in one go (continuously), the lagging strand is replicated in small pieces.
What is the role of DNA polymerase?
Has a proof-reading 3’ -> 5’ exonuclease activity.
When was the directionality of synthesis established? By who?
1968.
By Reiji and Tsunesko Okazaki.
There is a leading strand, synthesised continuously. Here 5’ -> 3’ synthesis proceeds in the same direction as the replication fork.
There is a lagging strand, synthesised discontinuously. Here 5’ -> 3’ synthesis proceeds in the opposite direction as the replication fork.
What is the purpose of Okazaki fragments?
So that synthesis of new DNA is in one direction only- new DNA is built in the 5’ -> 3’ direction.
Why is there no 3’ to 5’ synthesis of new DNA?
Hypothetical: misincorporation, proofreading: one nucleotide removed, leaving phosphate but a 5’ triphosphate is required here. No 5’ triphosphate available for hydrolysis: no energy for polymerisation.
Reality: Misincorporation, proofreading: one nucleotide removed, leaving a 3’ OH. Incorporation of correct nucleotide and chain extension. Hydrolysis of the incoming nucleotide provides the energy for polymerisation.
5’ -> 3’ synthesis allows editing, whereas 3’ -> 5’ does not.
What is the life cycle of M13?
M13- single stranded DNA genome.
Wrapped in a protein coat. Infected via the F pilus, which allows the exchange of genetic material. ssDNA genome is then replicated, now double stranded. Continues to replicate to form a ‘rolling circle’ of replication, to form single stranded genomes, then packaged out. Monitored by the molecular weight, using radioactive phosphate.
What did Arthur Kornberg and Tsunesko Okazaki discover about how DNA replication begins?
- Arthur Korberg- the replication of the M13 phage DNA from single-stranded infective form to double-stranded replicative form by an E. coli extract is prevented by rifampicin. Need to have a control, usually an inhibitor, but added an extra control by adding rifampicin (inhibitor of E coli RNA polymerase)- would expect nothing to happen.
Tsuneko Okazaki- found that DNase cannot completely destroy Okazaki fragments. It left little pieces of RNA, 10-12 bases long.
In vivo, the primer for an Okazaki fragment is RNA, not DNA.
How is RNA primer synthesised?
DNA primase is a rifampicin-sensitive DNA-directed RNA polymerase. It synthesises an RNA primer to initiate DNA synthesis on the lagging strand.
RNA polymerase does not require a primer.
How is the lagging strand synthesised?
Primers are anti-parallel to the DNA strand. RNA stretch at the beginning, followed by DNA.
- Lay down the primers. New RNA primers are synthesised by DNA primase.
- Extend the primers. DNA Pol III extends the RNA primer using dNTP’s to make Okazaki fragments on the lagging strands. As the replication fork separates more DNA, new primers are laid down by DNA primase.
- The extension crashes into the previous Okazaki fragment and RNA is erase. The old primers are erased by the 5’ -> 3’ exonuclease activity of Pol I and are replaced with new DNA.
- Seal the gaps by DNA ligase. Okazaki fragment is joined to the growing chain by DNA ligase sealing the gap.
How are Okazaki fragments joined?
DNA ligase uses ATP as an energy source, releasing pyrophosphate and attaching AMP to the 5’ phosphate of the downstream fragment.
AMP is released and the phosphodiester bond is formed between the 3’ OH of the upstream Okazaki fragment and the 5’ phosphate of the downstream fragment.
How does DNA polymerase move? Why?
In a loop.
Becuase the two Pol III molecules are held together, so the DNA strands must be facing the same way. Rather than the two Pol IIIs moving in opposite directions.
What does DNA replication involve?
Two Pol III attached by a clamp, with helicase and DNA primase.
DNA Pol I, with DNA ligase.
Single-stranded DNA binding protein.
What happens to the leading strand?
DNA helicase unwinds the DNA helix, separating the strand.
DNA primase manufactures an RNA primer on the leading strand template.
The primed duplex is captured by Pol III.
Pol III has a low processivity- it can only make short stretched of DNA before it falls of the DNA.
New clamp halves maintain the clamp holder in a state of readiness.
The clamp holder transfers the two halves of the beta clamp to Pol III- clamping converts Pol III to high processivity: it can now replicate long stretches of DNA.
Helicase continues to unwind, and Pol III replicates the leading strand continuously.
What happens to the lagging strand?
DNA primase manufactures an RNA primer on the lagging strand template.
The primed duplex is captured by Pol III and clamped- this forces the lagging strand into a loop. Each primed duplex has the 5’ end of the RNA primer facing away from the replication fork.
The helicase continues to unwind, Pol III replicates the leading strand continuously and extends the new primer on the lagging strand, until the old Okazaki fragment has been pulled back to Pol III.
New DNA crashes into an old RNA primer- this stimulates the unclamping. DNA Pol III has to have a low processivity in order to unclamp, otherwise could not release the new fragment as easily.
DNA Pol I and DNA ligase repair the gap.
DNA primase primes the lagging strand template and the process restarts by clamping the new lagging strand primer. DNA Pol I does not require high processivity.
What is processivity?
A measure of an enzyme’s ability to catalyse consecutive reactions without releasing its substrate.
When clamped, DNA Pol III can replicate a new strand at around 1000 bases per second.
What happens in Eukaryotic DNA replication?
Eukaryotic DNA Pols polymerise at ~50 nucleotides/s. 20x more slowly than E. coli Pol III.
It would take ~50 hours to replicate an average human chromosome at this rate.
DNA replication in eukaryotes proceeds bi-directionally from multiple origins of replication.
Bacteria and Eukaryote shave remarkably similar complex (same process/mechanism).
What is SSB?
Single stranded DNA binding protein.
Stem-loop structures can be formed if DNA is denatured and fails to re-anneal properly.
The same is true for single-stranded DNA.
DNA replication requires a supporting cast of SSB.
Prevents inappropriate base pairing in ssDNA during replication.
Protects ssDNA from base pairing and from nuclease. It is constantly being displaced by Pol III and being replaced as the helix is unwound.
What is the difference between positive and negative supercoiling?
Positive- results from overwinding of DNA.
So can occur upstream of the replication fork. Makes it harder to separate strands, compared to negative supercoiling.
Negative- results from the unwinding/underwinding of DNA. (Most DNA is negatively supercoiled).
Negative supercoiled DNA is easier to be replicated.
So topoisomerases are used to regulate the degree and type of supercoiling.
What is the role of topoisomerase?
Relaxes DNA.
What does Type II topoisomerase do?
(Gyrase in bacteria).
Convert overwound positively supercoiled DNA into underwound negatively supercoiled DNA.
Topo II binds the positive supercoil, makes a nick in both DNA strands, passes the DNA loop through the break and re-ligates it, forms a negative supercoil. Then the type II disengages.
What does Type I topoisomerase do?
Relaxes negatively supercoiled DNA.
Topo I binds the negative supercoil, makes a nick in one DNA strand, unwinds the DNA and re-ligates it, results in relaxed DNA.
What direction is bacterial DNA polymerisation?
Two Pol III complexes enter the DNA at an origin of replication.
Replication proceeds in both directions at the same time.
When the two forks meet, topo IV (a type of topo II) separates the catenated daughter chromosomes by a double stranded break and relegation.
Moves from left to right, has a leading strand and a lagging strand. Ends up with two linked chromosomes- catenated. So E. coli can dive without ripping its DNA into two pieces.
What are telomeres?
Built-in ageing mechanism.
Two of them for every one of your haploid chromosome.
Why are telomeres a major problem for eukaryotes?
The lagging strand template can be primed at or near the telomere (and then extended).
The DNA polymerase complex falls off.
The primers are erased.
This gap is filled by DNA polymerase and repaired by a DNA ligase.
This gap on the lagging strand cannot be filled by a DNA polymerase as there is no primer.
What happens to telomeres after every cell division/replication?
They get shorter (as do chromosomes).
It is an inbuilt ageing mechanism that stops cells dividing forever.
Some cells express telomerase that extend telomeres.
What is the correlation between telomeres and ageing/cancer?
Telomere length has a correlation with cellular ageing.
Telomerase is active in eggs, sperms, germline cells, epithelial cells, haematopoietic cells and other cancer cell lines.
Telomerase is responsible for the immoral phenotype of cancer cells.
What is the role of reverse transcriptase?
Provides an RNA template to synthesise a DNA copy of the template at the 3’ end of the parental lagging strand template.
This result is that telomeres are built of repetitive motifs.
