DNA Synthesis Flashcards
Why is DNA synthesis semi-conservative?
Because the helix is unwound, the strands are separated and each of these strands are used to make two duplexes hence in each strand is semi-conserved
Where is DNA replication initiated?
At the replication origins
How does DNA replication start?
The replication origins are recognised by an initiation complex
DNA at the origin unwinds to form a replication bubble and allows access to the replication machinery, the DNA polymerase and other factors
DNA synthesis occurs in a specific phase (S) of the cell cycle and involves complete unwinding of the parental DNA
How long does the bacterial cell cycle last?
20 to 30 minutes
How long does the mammalian cell cycle last?
16 to 24 hours
What happens in the M phase?
Mitosis where the chromosomes are compacted and separated to each of the daughter cells
What happens in the G1 phase?
In Gap 1, the cell is preparing to synthesise its DNA, so it is making all the histones and other proteins necessary for the next phase
What happens during the S phase?
During the synthesis phase, the DNA is synthesised wow
What happens during the G2 phase?
During the Gap 2 phase, the cell is preparing to condense the DNA in preparation for mitosis
What does each of the bacterial DNA polymerases do?
DNA polymerase I and II are for repair
DNA polymerase III is for replication
Pol I is too slow to replicate bacteria and actually mutants of E.coli were found with inactivated Pol I and yet were able to grow through DNA replication
What does each of the eukaryotic DNA polymerases do?
Alpha- replication Beta- repair Gamma- replicates mitochondrion DNA Delta- replication Epsilon- replication Others repair & translesion DNA synthesis meaning to say that when the replication fork encounters some kind of block due to a chemical reaction of a mutagen etc. they are able to work around the lesion and allow DNA replication to continue
What are the key properties of DNA polymerase?
Acts in 5’ to 3’ direction which has important consequences
Utilises A-T and C-G base pairing to synthesise new DNA strand
Requires a DNA template, a DNA or RNA primer ( a small piece of DNA or RNA which is synthesised and then annealed to the template DNA strand providing a substrate for the DNA Pol, the 3’ OH end), the four deoxyribonucleoside triphosphate (dNTP) building blocks and Mg2+ ions
Has a proof-reading function
Recall the process of DNA synthesis
- DNA Pol brings the appropriate dNTP into the active site and positions it next to the end of the growing newly synthesised strand
- DNA polymerase finds and pairs the complementary base to the one on the template strand
- Once the dNTP has been base-paired to the template strand, this allows 3’ OH end of the newly synthesised strand to attack the inner-most phosphate of the triphosphate group
- This forms a 3’-5’ phosphodiester bond and it displaces pyrophosphate (P2O7) from the building block
- The pyrophosphate is destroyed; hydrolysed to phosphate molecules by an enzyme called pyrophosphatase
- The dNTP now has a 3’ Oh end (this explains the directionality)
What is the lagging strand?
The lagging strand is the strand replicated discontinuously
The problem with the lagging strand is that as the replication fork moves away the polymerase is copying DNA in the direction opposite to that of the movement of the replication fork
This results in the DNA being synthesised in small fragments called Okazaki fragments (150- 200base pairs)
How is the lagging strand synthesised in eukaryotic cells?
- DNA helicase uses ATP to break the hydrogen bonds between the base pairs
- Polymerase alpha puts down a small amount of RNA and then switches to synthesising a little bit of DNA
- The DNA can be used as a primer to allow the replicative polymerase to come in the synthesise and DNA
- These Okazaki fragments all start with a little bit of RNA then DNA is present for the rest of the fragment
TO join all these fragments you have to take out the RNA - DNA ligase links all of the Okazaki fragments together
