*semi Conservative Replication

Question 1

Explain how meselson and stahl found semi conservative replication

Answer 1

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

Question 2

How does hybrid dna of different isotopes show semi conservative and not conservative

Answer 2

Conservative would have only N15 strands OR N14 not both

Question 3

What does dna replication firstly require to produce the new strands

Answer 3

DNTPs

Base, sugar and 3 phosphates

Question 4

What is the reaction DNA polymerase catalyses

Answer 4

DNA + DNTPs

To

DNA+1 nucleotide and PPi (pytophosphate)

Question 5

What is DNA polymerases III job in dna replication

Answer 5

To catalyse the formation of phosphodiester bonds on the new strands (DNTPs) between the C3OH and a phosphate

Question 6

Why is a primer required to form new strands

Answer 6

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

Question 7

Where is replication initiated from and how ?

Answer 7

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.

Question 8

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

Answer 8

Uses ATPase enzyme (needs atp to break H bonds)

Question 9

What proteins are recruited to the broken dna strands after helicase ?

Answer 9

Single stranded binding proteins (SSB)

Bind to both single strands when broken by dnaB helicase

Question 10

What is the importance of SSB proteins ?

Answer 10

Prevents the single strands forming secondary structures via base pairs

Question 11

What are the groups of enzymes found at the wound dna before helicase breaks them?

Answer 11

Topoisomerases

Question 12

What is the importance of topoisomerases on wound dna

And what’s the difference between I and II

Answer 12

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

Question 13

What is the enzyme that synthesis rna primers for strand replication initiation by DNA polymerase III

Answer 13

Primase (produces primers for both the lagging and leading strand)

Question 14

What is primase bound to during replication

Answer 14

DnaB helicase.

Question 15

Does replication start after all of DNa is unwound or straight after dna helicase breaks the bonds?

Answer 15

Straight after

On both the leading and lagging strand

Question 16

What are the 3 main parts of the DNA polymerase III core

Answer 16

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

Question 17

The alpha subunit forms a cleft like structure. What is this important for when catalysing formation of new dna

Answer 17

Allows the right complementary base pairing because the shape only allows for them to be complementary

Question 18

If bases on DNTPs aren’t complementary, how is this mistake eradicated

Answer 18

Through the exonuclease domain on the DNA polymerase III which removes the base which isn’t correct

Question 19

What is the leading and lagging strand

Answer 19

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

Question 20

Because DNA polymerase III can only synthesise from 5’ to 3’ , what does this form on the lagging 3’ to 5’ strand?

Answer 20

Okazaki fragments - new dna strands are produced in fragments instead of continuous

All have different primers

Question 21

What is the DNA polymerase III holoenzyme complex made of

Answer 21

DnaB helicase
Primase
SSB proteins
2 DNA polymerases for coordinated synthesis in same direction (5’ to 3’

Question 22

When does the loop on the lagging strand get bigger?

Answer 22

When more unwinding from helicase occurs and when new DNA is synthesised from beginning of primer

Question 23

Why do okazaki fragments all have new primers attached to them when they are formed on the lagging strand

Answer 23

Because every time new single stranded dna is unwound primers need to be added for the synthesis of the new strand in the loop

Question 24

What is DNA polymerase I job in the filling of Okazaki fragments?

Answer 24

It displaces the rna primers first and replace it with DNA fragments

Question 25

What does DNA Ligase do when the primers from okazaki fragments have been removed?

Answer 25

They catalyse the phosphodiester bond formation between the different fragments so it’s a continuous new strand of DNA