Lecture 4 Flashcards
Who were the first scientists to show how DNA replicated and how did they do so?
Meselson and Stahl (1958)
Used E.coli cells and grew them in the presence of 15N (heavier than normal version 14N)
Therefore all DNA nitrogen became 15N, made up of 2 heavy strands
This was then grown in the presence of 14N for a generation
DNA then underwent density gradient centrifugation
Results were as followed:
Generation 0 - HH high density
Generation 1 - LH intermediate density
Generation 2 - LH + LL intermediate and low density
What is DNA replication?
Duplication of all the DNA in the cell
Each strand of a DNA helix is a template for synthesis of a new strand
Each new DNA helix contains - 1 template strand of DNA and one newly synthesised strand of DNA
What are the origins of replication?
Unique sites on the DNA helix where replication begins
What are replication forks?
Site on the DNA helix where active replication occurs
Shifts along DNA as it is replicated
What does bi-directional replication mean?
Both directions from a single origin
Each new strand of DNA synthesised 5’->3’
Each template strand copied 3’->5’
How does prokaryotic DNA replication compare to eukaryotic replication?
Same basic mechanism
Chromosome topology differemt (prokaryote has circular DNA eukaryotes have linear DNA)
Proteins involved in replication are related structurally and functionally although there are many more varieties in eukaryotes
Describe the replication of E.coli
Circular DNA
- origin - point in which replication starts, complex region where replication and control of growth cycle are regulated
- replication fork - point at which DNA strands separate and synthesis of new DNA begins, 2 replication forks proceed bi directionally away from the origin
- termini - replication of circular DNA terminates 180 degrees from the origin
What is the name given to a piece of DNA which replicates as a single unit?
A replicon
What is the difference between eukaryotic and prokaryotic replicons?
Prokaryotes have a single origin of replication while eukaryotes have multiple origins of replication
Describe the DNA replication of a eukaryote
Has several origins - simpler than prokaryote origins
- Activated in the S-phase of the cell cycle
- origins tend to be AT rich as this is easier to open
Replication fork - 2 forks proceed bi directionally from each origin
Termini - replication bubbles fuse to form the complete DNA
Synthesis of both strands is simultaneous - double stranded parent DNA is separated and both strands act as a template for the synthesis of 2 new strands
What is the problem with the synthesis of both strands in eukaryotic DNA replication?
The enzyme that catalyses DNA synthesis works exclusively in the 5’->3’ direction and strands are anti-parallel
What is the leading strand?
The parental DNA strand that runs in the 3’->5’ direction
New strand is synthesised continuously in the 5’->3’ direction
Phosphodiester bonds are formed between the 3’ OH and the 5’-phosphate such that the chain always has a free 5’-phosphate and a free 3’-OH
What is the lagging strand?
The parental DNA running 5’->3’ direction
New DNA strand is synthesised discontinuously
Synthesis proceeds from the replication fork in the 5’->3’ direction back towards the origin
In a discontinuous fashion short fragments are synthesised - 1000-2000 nucleotides in prokaryotes 100-200 nucleotides in eukaryotes
What are the short fragments known as in lagging strands?
Okazaki fragments
