Dna Replication Flashcards
What are the three proposed models of replication
- Conservative replication
- Dispersive replication
- Semiconservative replication
Conservative replication
One daughter cell inherits two of the old strands, and the other daughter cell inherits both new strands
Dispersive replication
The new standard contains both the new and old dna (the original strands don’t exist anymore)
Each daughter cell inherits mixed strands
Semiconservative replication
Both daughter molecules inherit one old and a new strand
Messelson stall experiment
To determine which model of replication applies to nature
Used heavy nonradioctive n14 to get bacteria that had “heavy” dna
Bacteria 2ith only heavy dna was placed into a medium containing normal weight n14 and isolated them after 1, 2, etc. generations
The results of the experiment show that semiconservative replication is the correct model
Dna synthesis in E.coli
- dsDNA is separated, and the replication bubble is formed
- Dna helicase binds to lagging strands and travels towards replication fork -> creating ssDNA that are held together by single strand binding proteins (SSB) to increase stability
- DNA gyrase and DNA topoisomerase I reduce supercoiling stress right in front of the replication fork by cutting dna strands , letting them rotate and bidning them back together
Leading strand replication
- Dna primase makes primer to which dna pol III can attach and start to elongate the dna
- Dna synthesis on the leading strand is continuous. Primers are removed by dna pol I and ligase joins the ends together (between rest of newly synthesised strand and replaced primer)
What happens when nucleotides are added to the growing chain
Activated triphosphate forms from the deoxynucleotides that are added to the free 3’ end of the chain.
Free 3’OH attacks the alpha phosphate of the dNTP releasing pyrophosphate (ppi) which is further hydrolyzed to 2 phosphates (by pyrophosphatase)
Lagging strand replication
- Dna synthesis is not continuous and happens in fragments instead it creates okazaki fragments
- Dna primase makes a primer from which dna pol III synthesises the strand. Once dna pol III meets a new primer, it detaches from the lagging strand and attaches to the next primer to continue synthesis
- Away from the replication from -> reason why dna pol III has to always go back to the new primers near the replication fork to continue
- Dna pol I also has exonuclease activities, meaning that it also goes from 3’ to 5’ and remove3s the primers and replaces them with dna nucleotides
- Dna ligase then basically glues the gaps together using atp as a cofactor
Coupling of the two strands during replication
The leading and lagging strands are coupled. Both dna pol III are connected to a complex called the DNA pol III holoenzyme or repliosome
Repliosome = Complex of helicase, binding proteins, polymerase, clamp proteins
This linking facilitates coordination synthesis and efficient reinitiation dna on the lagging strand
Clamp protein increases the porcessivity of dna pol III by making sure it doesn’t fall off the strands
Dna primase is attached to the helicase, which ensures that primers are only made on the lagging strand right next to the replication fork
Replication in eukaryotes
Similar to prokaryotes:
1. Origin of replication
2. Primers
3. Dna pol delta
4. Ligase
5. Accessory proteins
6. Clamp protein
Difference:
Removal of rna primers occurs, but we don’t know through which mechanism
Initiation in prokaryotes
- Initiated at origin of replication (oriC) where a sequence is present that is specifically recognised by initiator protein DnaA
- DnaA binds to oriC, which distorts the double helix and starts to unwind
- DnaA recruits helicase DnaB to unwind Dna and start replication
Initiation in eukaryotes
- There are 30-50k origins of replications in humans, and it has to be ensured that they only fire once
- Origin of replication are licensed by ORC, Cdc6, Cdt1, Mcm2-7 complex
- Cdc6 and Cdt 1 are synthesised in G1 and destroyed in S phase
- Thus, the origin of replication can only be licensed in G1
Adding nucleosomes to Dna during dna replication (eukaryotic)
Because both strands have the same density of nucleosomes as the parental dna, nucleosomes are added during replication
Nucleosomes are loaded onto the dna via CAF-1 and through that, it brings histone directly brings histonesnto the replication fork
The new nucleosomes contained a mix of old and new histone and disassemble during replication
End replication problem in eukaryotes
Problem:
Genome is linear, and removal of the primers at the end of the dna strand leaves a gap because dna pol I cannot attach a 5’ end to the free 3’ OH -> technically the genome should get shorter every replication cycle
Solution:
Telomeres are extensions at the ends of the dna strands that consist of simple guanine rich repeats (telomeric repeats) thus Telomers are lost at the end of replication and not vital dna sequences. Telomeres are lengthened through telomerase, an enzyme that can base pair small rna templates to free 3’ end and add nucleotides to it
