Lecture 1

Question 1

What is a model organism?

Answer 1

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.

Question 2

Nomenclature of genes and proteins

Answer 2

dnaA: gene (lower case first letter and italics)
dnaA-: mutation
DnaA: protein (first letter upper case and no italics)

Question 3

What are the three models for DNA replication?

Answer 3

Semi-conservative
Coservative
Dispersive

Question 4

Semi-conservative replication

Answer 4

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.

Question 5

Conservative replication

Answer 5

The parental molecules directs synthesis of an entirely new double stranded molecule, after one round of replication one molecule is conserved as two strands.

Question 6

Dispersive model

Answer 6

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.

Question 7

What proved the semi-conservative model?

Answer 7

Meselson-Stahl experiment

Question 8

Describe the Meselson-Stahl experiment

Answer 8

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.

Question 9

Explain the results of the Meselson-Stahl experiment

Answer 9

Start of experiment: all DNA was heavy

One generation: half of the DNA was intermediate and half was light

Question 10

Who was involved in the discovery of the replication forks?

Answer 10

Cairns

Prescott & Kuempel

Question 11

3 step experiment of identifying the 2 bi-directional forks

Answer 11

Grow E.coli in media without radiolabelled 3H- thymidine
Grow 3H- thymidine of low specific activity
Grow 3H- thymidine of high specific activity

Question 12

What type of replication occurs at the forks?

Answer 12

Continuous replication in the 5’ to 3’ direction

Discontinuous replication in the 3’ to 5’ direction which forms okazaki fragments

Question 13

Who showed that DNA replication is a discontinuous process?

Answer 13

Sakabe & Okazaki

Question 14

Why is replication discontinuous?

Answer 14

DNA polymerase copies only in the 5’ to 3’ direction

Question 15

Theta replication

Answer 15

Electron micrographs show replication of circular plasmid DNA

Question 16

Who purified and characterised the first DNA copying enzyme?

Answer 16

Kornberg

DNA polymerase I

Question 17

What does DNA polymerase require to function?

Answer 17

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

Question 18

Function of a primer

Answer 18

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.

Question 19

How frequently does DNA polymerase make mistakes and can it correct these?

Answer 19

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

Question 20

What events would follow Polymerase incorrectly pairing a C with T

Answer 20

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

Question 21

What is used to prime DNA synthesis & why?

Answer 21

A short segment of RNA as synthesised by a DNA polymerase

RNA synthesis does not require a prime

Question 22

Briefly outline replication in 4 steps

Answer 22

1. DNA replication is primed by RNA. The RNA primer is synthesised by DNA primase
2. DNA polymerase extends from the primer
3. The process is repeated when Okazaki fragments are being formeed. A new primer is needed for each Okazaki fragment
4. DNA polymerase anneals to the 3’ OH to synthesise DNA

Question 23

Primer location for the leading and lagging strand

Answer 23

The leading strand is primed once at the origin of replication.
The lagging strand has to be primed separately for each Okazaki fragment

Question 24

Dna G

Answer 24

Primase enzyme in E.coli

Synthesises DNA primers that are 10-12 nucleotide long oligoribonucleotides

Question 25

DNA ligase

Answer 25

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

Question 26

DNA helicase

Answer 26

Strands are unwound by DNA helicase that transcerses along single stranded DNA

Question 27

DnaB

Answer 27

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)

Question 28

DNA polymerase I and II

Answer 28

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

Question 29

Summarise the start of semi-conservative replication

Answer 29

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.