Lecture 1 Flashcards
What is a model organism?
A non-human species that is extensively studied to understand particular biological phenomena with the expectation that discoveries made in the organism model will provide insight into the workings of other organisms.
Nomenclature of genes and proteins
dnaA: gene (lower case first letter and italics)
dnaA-: mutation
DnaA: protein (first letter upper case and no italics)
What are the three models for DNA replication?
Semi-conservative
Coservative
Dispersive
Semi-conservative replication
Two parental strands separate and each makes a copy of itself. After one round of replication the two daughter molecules each contain one old and one new strand.
Conservative replication
The parental molecules directs synthesis of an entirely new double stranded molecule, after one round of replication one molecule is conserved as two strands.
Dispersive model
Material in two parental strands is distributed more or less randomly between two daughter molecules. Old material could be distributed symmetrically or asymetrically between daughter cells.
What proved the semi-conservative model?
Meselson-Stahl experiment
Describe the Meselson-Stahl experiment
E.coli was grown in heavy 15N then transferred to grouw in normal light and 14N conditions
DNA was extracted from the bacteria and centrifuged to equilibrium in caesium chloride (CsCl)
A gradient was established so molecules would separate according to their density
DNA containing large amounts of heavy nitrogen 15N would sink lower.
Explain the results of the Meselson-Stahl experiment
Start of experiment: all DNA was heavy
One generation: half of the DNA was intermediate and half was light
Who was involved in the discovery of the replication forks?
Cairns
Prescott & Kuempel
3 step experiment of identifying the 2 bi-directional forks
Grow E.coli in media without radiolabelled 3H- thymidine
Grow 3H- thymidine of low specific activity
Grow 3H- thymidine of high specific activity
What type of replication occurs at the forks?
Continuous replication in the 5’ to 3’ direction
Discontinuous replication in the 3’ to 5’ direction which forms okazaki fragments
Who showed that DNA replication is a discontinuous process?
Sakabe & Okazaki
Why is replication discontinuous?
DNA polymerase copies only in the 5’ to 3’ direction
Theta replication
Electron micrographs show replication of circular plasmid DNA
Who purified and characterised the first DNA copying enzyme?
Kornberg
DNA polymerase I
What does DNA polymerase require to function?
Template strand to copy
Primer (another strand annealed to the template to supply a 3’ OH group)
dNTPs: deoxynucleoside triphosphates
Adds nucleotides to the 3’ end of the growing strand
Function of a primer
Another strand that is annealed to a template to supply a 3’ OH group
The primer starts synthesis and creates an insertion site where nucleotides can be added so the chain extends.
How frequently does DNA polymerase make mistakes and can it correct these?
Mistake every 1 in 5 X 10^7 bp
DNA polymerase has the capacity to correct errors using the 3’-5’ exonuclease activity that can remove incorrectly inserted nucleotides
PROOF READING FUNCTION PROVIDED BY AN EXONUCLEASE SITE
What events would follow Polymerase incorrectly pairing a C with T
Polymerase repositions the incorrectly paired 3’ terminus iinto the 3’ - 5’ exonuclease site
An exonuclease hydrolyses the mis-paired C
The 3’ terminus repositions
Polymerase incorporates the correct nucleotide
What is used to prime DNA synthesis & why?
A short segment of RNA as synthesised by a DNA polymerase
RNA synthesis does not require a prime
Briefly outline replication in 4 steps
- DNA replication is primed by RNA. The RNA primer is synthesised by DNA primase
- DNA polymerase extends from the primer
- The process is repeated when Okazaki fragments are being formeed. A new primer is needed for each Okazaki fragment
- DNA polymerase anneals to the 3’ OH to synthesise DNA
Primer location for the leading and lagging strand
The leading strand is primed once at the origin of replication.
The lagging strand has to be primed separately for each Okazaki fragment
Dna G
Primase enzyme in E.coli
Synthesises DNA primers that are 10-12 nucleotide long oligoribonucleotides
DNA ligase
Closes the nicks in the DNA backbone between Okazaki fragments
The strand is ligated by catalysing the formation of a phosphodiester bond
NAD is used as a cofactor for this reaction
DNA helicase
Strands are unwound by DNA helicase that transcerses along single stranded DNA
DnaB
5’3’ helicase which unwinds the parental DNA as it moves
Homohexamer (6 identical subunits) that forms a ring and has high processivity (rarely galls off)
DNA polymerase I and II
DNA polymerase II is the replicative polymerase in E.coli
DNA polymerase I can add 10-20 nucleotides per second and has a low preocessivity so will fall off after synthesising 3- 200 nucleotides
Summarise the start of semi-conservative replication
At this replication site DNA synthesis starts at a short RNA primer synthesised by DnaG primase. Two bidirectional replication forks are formed each with a leading and lagging strand. The leading strand of the DNA is synthesised continuously, and the lagging strand is synthesised discontinuously by short RNA primed Okazaki fragments. DnaB helicase unwinds the parental duplex allowing assembly of the DNA replication proteins.
