DNA replication

Question 1

What are 2 features of DNA replication?

What’s the difference in number of origin of replications between eukaryotes & bacteria?

What is the origin of replication composed of?

How is it more exposed?

what are the 3 steps for opening up the origin of replication?

what do each of the 3 proteins do?

Answer 1

semi-conservative
bidirectional - 1 origin of replication & 2 growing forks

Eukaryotes = lots
Bacteria = only 1 origin

9mers and 13mers

relaxation of negatively supercoiled template

1. DnaA recognises 9mers on DNA- causing them to bind & coil leaving 13 mers exposed
2. hydrolysis of 1 ATP releases energy so 13mer region opens up
3. hydrolysis 1 ATP brings DnaB & DnaC to further separate the DNA strand to form a prepriming complex for replication

DnaA = DNA binding protein (to 9mers)
DnaB = helicase
DnaC = helicase loader

Question 2

what are the x steps for DNA replication after separating the DNA at the origin of replication?

what is the DNA polymerase III holoenzyme made up of and what is it for? (3 bits)

what is the topoisomerase function? what are the 2 different types?

how does DNA replication occur on the lagging strand?

Answer 2

1. RNA primer is made by primase at origin of replication (in 5’ to 3’ on template/3’ to 5’ on coding)
2. DNA polymerase III binds to RNA primer & moves 5’ to 3’ of template to make daughter strand
3. continues until DNA polymerase III meets next origin of replication (or in bacteria completes cycle)
4. DNA polymerase I degrades RNA primer & DNA ligase seals gaps in DNA = continuous strand DNA

core enzyme/body = polymerisation
flexible linker proteins = to access DNA
sliding clamp & loader = load clamp onto DNA & stay fastened along strand

maintains helical density by relaxing part of the DNA for replication- negatively supercoils the origin of replication causing positive supercoiling of rest of DNA
Type I = relaxes DNA by nicking & closing 1 strand of duplex DNA
Type II = changes DNA topology by breaking/relaxing & rejoining double strand DNA

forms Okazaki fragments which are eventually joined up by DNA ligase at the RNA primers (after their degradation by DNA polymerase I)

Question 3

what causes DNA replication accuracy to be high?

how does DNA polymerase proofread the daughter strand of DNA formed in DNA replication?

what is a deamination error?

what are the 2 types of repair?

what are the structural differences between NTP, dNTP & ddNTP?

Answer 3

DNA polymerase proof reading & post-polymerase repair systems

1. DNA helix separates into replication bubble in the palm
2. Fingers close, bringing DNA into closer proximity
3. if incorrect base pair, closing of fingers move DNA to exonuclease domain
4. incorrect base removed 3’ to 5’ exonuclease activity
5. hand opens & DNA growing strand moves back to palm domain

changing of certain groups on a base to form a different base- causing in mutant DNA

mismatch = before replication finishes
excision repair = after replication finishes

NTP = OH at 2' & 3'
dNTP = OH at 3'
ddNTP = H at 3'