*semi Conservative Replication Flashcards
Explain how meselson and stahl found semi conservative replication
They put dna of ecoli into N15 isotope medium for 1 replication and found high density equilibrium point (line on bottom)
Then they put it into N14 for another replication cycle. This however showed a density equilibrium point in the middle- showing hybridity
Then they put it in for more cycles and found overall more N14 isotope dna but the N15 was still present
How does hybrid dna of different isotopes show semi conservative and not conservative
Conservative would have only N15 strands OR N14 not both
What does dna replication firstly require to produce the new strands
DNTPs
Base, sugar and 3 phosphates
What is the reaction DNA polymerase catalyses
DNA + DNTPs
To
DNA+1 nucleotide and PPi (pytophosphate)
What is DNA polymerases III job in dna replication
To catalyse the formation of phosphodiester bonds on the new strands (DNTPs) between the C3OH and a phosphate
Why is a primer required to form new strands
The primer sequence has a C3’ OH exposed.
This is what allows nucleophillic attack to break PPi and for the formation of a new phosphodiester bond with new DNTPs
Where is replication initiated from and how ?
OriC site which is a sequence of 5 binding sites to a dnaA protein
Also has an AT rich sequence (easily broken)
Initiates replication when the dnaA binds to the oric binding sites
Unwinds the AT rich sequence
This forms the Replication bubble start.
DnaB helicase is recruited when the AT rich sequence starts to unwind at the oriC (multiple orics in eukaryotes). What does it use to unwind the 5’ to 3’ strand of DNA
Uses ATPase enzyme (needs atp to break H bonds)
What proteins are recruited to the broken dna strands after helicase ?
Single stranded binding proteins (SSB)
Bind to both single strands when broken by dnaB helicase
What is the importance of SSB proteins ?
Prevents the single strands forming secondary structures via base pairs
What are the groups of enzymes found at the wound dna before helicase breaks them?
Topoisomerases
What is the importance of topoisomerases on wound dna
And what’s the difference between I and II
Topoisomerase catalyse the untangling of dna
I - does this by cleaving 1 strand then resealing
II- cleaved both strands on 1 molecule and this allows passage of the other molecule through it then reseals the break
What is the enzyme that synthesis rna primers for strand replication initiation by DNA polymerase III
Primase (produces primers for both the lagging and leading strand)
What is primase bound to during replication
DnaB helicase.
Does replication start after all of DNa is unwound or straight after dna helicase breaks the bonds?
Straight after
On both the leading and lagging strand
What are the 3 main parts of the DNA polymerase III core
A subunit - catalyses the formation of phosphodiester bonds of new bases
B2 cleft - accommodates the double stranded DNA once gone through a subunit
E subunit - exonuclease domain
The alpha subunit forms a cleft like structure. What is this important for when catalysing formation of new dna
Allows the right complementary base pairing because the shape only allows for them to be complementary
If bases on DNTPs aren’t complementary, how is this mistake eradicated
Through the exonuclease domain on the DNA polymerase III which removes the base which isn’t correct
What is the leading and lagging strand
The leading strand is the growing strand from 5’ to 3’ on one of the single strands when helicase unwinds it
Lagging strand is synthesis from 3’ to 5’ to form a new strand on the other single strand
Because DNA polymerase III can only synthesise from 5’ to 3’ , what does this form on the lagging 3’ to 5’ strand?
Okazaki fragments - new dna strands are produced in fragments instead of continuous
All have different primers
What is the DNA polymerase III holoenzyme complex made of
DnaB helicase
Primase
SSB proteins
2 DNA polymerases for coordinated synthesis in same direction (5’ to 3’
When does the loop on the lagging strand get bigger?
When more unwinding from helicase occurs and when new DNA is synthesised from beginning of primer
Why do okazaki fragments all have new primers attached to them when they are formed on the lagging strand
Because every time new single stranded dna is unwound primers need to be added for the synthesis of the new strand in the loop
What is DNA polymerase I job in the filling of Okazaki fragments?
It displaces the rna primers first and replace it with DNA fragments
What does DNA Ligase do when the primers from okazaki fragments have been removed?
They catalyse the phosphodiester bond formation between the different fragments so it’s a continuous new strand of DNA
