Lecture 1 - DNA replication

Question 1

What are the broad phases of DNA replication?

Answer 1

Initiation - first phase - where all replication proteins bind near the origin and form a replication fork
Elongation - forks moving along genome and duplicating DNA
Termination - where replication forks are fusing

Question 2

What is the replicon model?

Answer 2

Particular sequence called replicator and that is what makes an origin an origin
Replicator sequence confers origin activity
Binds initiator protein
ORC name for replication origins in bacteria and DnaA initiator

Question 3

Describe how initiation takes place in E. coli

Answer 3

The DUE (duplex unwinding element) box is very AT rich - fewer hydrogen bonds so easier to unwind
First many DnaA bind to DnaA box sequences to make an oligomer, then many DnaC, the job of which is to load the replicative helicase, DnaB

Question 4

Describe how to carry out an ARS assay

Answer 4

Find out where origins are
Fragment genome and by chance some fragments will have origins in
Clone fragments into plasmid with a selectable marker and use to transform host cell
Host cell can’t survive unless you get expression of this selectable marker
If replicator sequence cloned in rather than random DNA then plasmid can replicate

Question 5

How can collisions be avoided in circular genomes?

Answer 5

Helicases travelling with the replication fork can push blockages out of the way
If RNA polymerase is stalled the transcript can be cut to reset transcription
Helicases can remove blockages

Question 6

What are the roles of topoisomerases?

Answer 6

Resolving supercoiling induced by transcription
Resolving supercoiling induced by replication
Separating linked chromosomes after DNA replication

Question 7

Explain the importance of telomeres

Answer 7

Linear chromosomes have an end and due to the directionality of DNA polymerase this poses a problem - gaps between Okazaki fragments which may contain important genes
To solve this problem eukaryotes don’t have genes at the very end of their chromosomes but repetitive sequences called telomeres. They also have an enzyme called telomerase which contains an RNA template that can bind whatever the particular repeat sequence is in that genome. Telomerase also has reverse transcriptase activity so when the RNA template binds it synthesises an RNA primer for DNA polymerase to complete the replication. This leaves a single stranded end that loops back into the duplex to form a T loop

Chromosome ends are left with a 3’ overhang

The protruding end loops back and the ssDNA end is inserted into the duplex = T-loop

T-loops mark chromosome ends

Question 8

What is the difference between bacterial and archaeal replication?

Answer 8

Order of DNA melting and helicase loading reversed