Mechanisms for DNA replication
There are three different hypotheses for the mechanism of DNA replication:
Taylor, Wood and Hughs
Taylor , Wood and Hughs did some early work on DNA replication, they used V.faba roots and H-dTTP.
This shed some light on the problem but it was not until Meselson and Stahl (1958) published their work with E.coli that the problem was solved.
Meselson and Stahl (1958)
Experiments with E.coli
- Cultured E.coli in brother containing 15NH4Cl
- Switched the broth to 14NH4Cl
- 14N-DNA vs 15N-DNA
- If the DNA samples are run on CsCl density gradient, 14N-DNA can be distinguished from 15N-DNA as they have different densities. 15N-DNA is heavier than 14N-DNA.
Starting with E.coli grown in brother containing 15NH4Cl (all dNA heavy):
- Switch broth to 14NH4Cl and take samples at 20 minute intervals
- 20 minutes = 1 life cycle = 1 round of DNA replication
- As the DNA replicates, the newly synthesised DNA will be 'light'
- Run DNA samples on CsCl and can distinguish between the three potential methods of DNA replication
Conclusion:- DNA replication is semi-conservative
Which enzyme is involved?
Kornberg et al (1957)
- worked with E.coli
- isolated DNA polymerase
- enzymology was not what was expected
- DNA polymerase was equally good as a nuclease as a polymerase
Cairns and De Lucia (1969)
- isolated E.coli mutant with only 1% of Kornberg's DNA polymerase activity but which was still able to divide effectively
- However it could repair DNA damaged by UV light
DNA polymerase I: a repair enzyme
There have been two other E.coli DNA polymerases found since the first one:
- DNA polymerase II
- DNA polymerase III
They only function 5' to 3' and will not work on ssDNA as they require a short dsDNA region to act as a primer.
Eukaryotic DNA Polymerase
- DNA replication takes place in the S phase
- There are give different DNA polymerases found mammalian cells:
- A (alpha) - replication of nuclear DNA
- B (beta) - DNA repair
- Y (gamma) - replication of mitochondrial DNA
- O (delta) - replication of nuclear DNA
- E (epsilon) - repair?
Mechanism for DNA replication
Mechanism described is for prokaryotes (E.coli) but similar mechanism is seen in eukaryotes.
- triphosphate deoxynucleotides dATP, dTTP, dCTP, dGTP
- DNA template + primer
Origin of Replication
- specific DNA sequences
- 1 per plasmid/bacterial genome
- eukaryotes have larger genomes so 1 per chromosome would be too slow
- several per chromosome
- yeast: 1 every 40kb
- mammals: 1 every 150kb
- seen in viral genomes
- REPLICON: any piece of DNA that replicates as a single unit
At replication, the sequence at the origin becomes single stranded. Once the origin is opened, DNA synthesis is bi-directional.
DNA synthesis from two origins
- Starting point - parent DNA strands and an origin of replication identified.
- Origin is open.
- DNA synthesis starts to generate daughter strands.
- Another origin of replication is identified.
- DNA synthesis has progressed at both origins of replication
- DNA replication that started at one origin of replication has met that progressing from the other origin.
Method for replication of DNA (E.coli)
- Both strands of DNA are synthesised simultaneously at each replication fork
- But the replication has to be 5' to 3'
- Since strands are antiparallel, how is the strand that runs 5'-3' past the replication fork copied?
Okazaki et al (1968)
- E.coli + 3H-dTTP incubated for a few seconds
- extracted DNA
- newly synthesised DNA will have incorporated the 3H-dTTP
- isolated small single stranded 3H-DNA fragments ~1000-2000 nt
- If the experiment was extended
- E.coli + pulse 3H-dTTP then dTTP
- the 3H-small fragments are incorporated into high MW DNA
These fragmens were called OKAZAKI FRAGMENTS
- prokaryotes 1000-2000 nt
- eukaryotes 100-200 nt
Semi-Discontinuous Replication Model
- Leading strand 5' to 3' continuously synthesised
- Lagging strand5' to 3' discontinuously synthesised
- Series of Okazaki fragments joined together later
Problems still outstanding:
- how the primer is set up
- how to deal with supercoiling/relaxed state as necessary
- how to maintain ssDNA as required
Range of Enzymes and Proteins
- single stranded binding proteins: SSBs
- bind to ** bubble at the origin of replication
- breaks the H bonds between the bps. ATP mediated
- migrates along the parental DNA molecule with the replication fork
- DNA gyrase (topoisomerase II)
- mediates unwinding of the dNA in advance of the replication fork
- primosome (RNA polymerase complex) + rNTP
- displaces SSBs and catalysed formation of RNA primer complementary to the DNA
- about 6 nt long
- replicase (DNA polymerase III) + dNTP
- replaces primosome, uses RNA as primer
- DNA polymerase I
- removes RNA primer and fills in the gap
- DNA ligase
- joins together any nucleotides as nece**ary
Primer production and Leading Strand Synthesis*
- DNA duplex opened at the origin of replication
- Helicase and DNA gyrase activity
- SSBs keep ssDNA as single strand
- Primosome (RNA polymerase) bind, synthessis complementary RNA molecule = primer
- SSBs displace
- Further unwinding of parental duplex at replication fork
- Primosome replaced by replicase = DNA polymerase III
- Synthesises complementary DNA molecule
- Furhter unwinding of parental duplex at replication fork
- Extension of the leading strand
Lagging strand synthesised in short bursts:
- RNA primer, then DNA
- RNA removed and gap filled by DNA polymerase I
- Strand joined by ligase
Fidelity of Replication and Mutations
Fidelity of Replication
E.coli: 1 mistake in 10^10 bases incorporated during replication. This is kept low by:
- only allowin complementary base pairs to be incorporated
- proof reading (exonuclease associated with the polymerase)
- 'mismatched bases', gaps, breaks, T-T dimers
- DNA is scrutinised continuously, not just during replication.
- Mistakes are identified
- DNA polymerase I is brought in to remove and replace
- DNA ligase to join up
- Some repairs may fix a mutation (eg mismatched bases)
- More pressure put on the system; more mistakes are made