Nucleotides & Nucleic acids (lectures 15-18)

Question 1

Functions of DNA

Answer 1

1) genetic code
2) storage within the cell
3) accessibility for transcription
4) replication
5) meiosis
6) genome integrity

Question 2

Components of nucleic acids

Answer 2

Heterocyclic base
Sugar (ribose or deoxyribose)
Phosphate

Question 3

What is a nucleotide?

Answer 3

Base + sugar + phosphate

Question 4

What is a nucleoside?

Answer 4

Base + sugar

Question 5

What are the nucleosides in RNA?

Answer 5

Adenosine
Guanosine
Cytidine
Uridine

Question 6

What are the nucleotides in RNA?

Answer 6

Adenylate
Guanylate
Cytidylate
Uridylate

Question 7

What are the nucleosides in DNA?

Answer 7

Deoxyadenosine
Deoxyguanosine
Deoxycytidine
Thymidine

Question 8

What are the nucleotides in DNA?

Answer 8

Deoxyadenylate
Deoxyguanylate
Deoxycytidylate
Thymidylate

Question 9

Polymeric structure of DNA and RNA

Answer 9

DNA backbone is intrinsically negatively charged
• Easy to work with
• Wraps around positively charged histones = makes DNA more compact
• Prevents attack from nucleophiles

DNA > 100x more stable than RNA as the OH groups on the RNA lead to base-catalysed hydrolysis of the RNA backbone
• RNA needs to be more unstable to perform its function
• Want it all to naturally degrade

DNA = long term storage of information 
RNA = temporary transfer of information

Question 10

What are Chargaff’s rules?

Answer 10

1) For a particular species: [A] = [T] & [G] = [C]

2) [A] + [T] / [G] + [C] varies between organisms

Question 11

Watson-Crick model for DNA structure

Answer 11

2 antiparallel strands 
Right handed double helix 
Specific base pairing 
Bases stacked on the inside 
Backbone on the outside

Question 12

Double helix dimensions

Answer 12

One turn of helix = 3.4 nm
Rise per base = 0.34 nm
10 base pairs per turn
Helix diameter = 2 nm

Question 13

How is strength of base pairing different?

Answer 13

G-C has 3 hydrogen bonds so is slightly stronger

Question 14

Sugar phosphate backbone

Answer 14

Negatively charged
Makes it difficult to pack tightly
Targeted for non-specific DNA binding
Binding by positively charged proteins

Question 15

Why do we get major and minor grooves?

Answer 15

DNA molecule is asymmetrical
Backbones are closer together on one side leading to the minor groove
Further away on the other side leading to the major groove

Question 16

Major groove

Answer 16

Information-rich
Sequence-specific DNA binding
‘Read’ the sequence without unwinding
Exploited by transcription factors

Question 17

Minor groove

Answer 17

Information-poor
Infrequently used for sequence recognition
Binding typically alters DNA architecture

Question 18

Single stranded nucleic acid structures

Answer 18

Always attempts to satisfy Watson-Crick base paring

RNA molecule manages to form a slight double helix due to palindromic sequences in the stem loop

Question 19

How idid Meselson & Stahl prove DNA was semi-conservative using radioactive nitrogen isotopes?

Answer 19

Grew bacteria in nitrogen 15 containing medium (heavy nitrogen) so all DNA would be heavy DNA
Would then continue growing then in nitrogen 14 (light nitrogen)

Old strands = n15
New strands = n14

Density gradient equilibrium sedimentation
• Centrifugation
• As a molecule is centrifuged it will sediment when the buoyancy becomes equivalent to the centrifugal force

The more rounds of replication the more light fomrs of DNA
Will always have 2 intermediate species – one light strand & one heavy strand

Question 20

DNA replication

Answer 20

Requires deoxyribonucleotide triphosphate (dNTP) precursors
The new DNA chain is assembled on a pre-existing DNA template
Requires a primer to begin synthesis
Mistake correction by the removal of mismatched nucleotides
• 3’-5’ exonuclease activity permits removal of incorrect nucleotides
• DNA polymerase travels 5-3 but it has the ability to reverse and & correct the mistake

Question 21

Whats the different between exonucleases & endonucleases?

Answer 21

Exo digest from the end

Endo digest from the middle

Question 22

Initiation of replication

Answer 22

Origin of replication (oriC)
One circular piece of DNA
DnaA is an oligomeric protein that can form rings or filaments
• Forms a 6 membered ring in bacteria
• 5 components of the ring bind to the 5 dnaA binding sequences
• AT rich tandem array of 13mers
• DnaA assembly stimulates unwinding of AT rich array

Question 23

Unwinding the double helix

Answer 23

DnaB helicase – unwinds DNA
• Ring structure with a hole in the middle – the right size for ssDNA but too small for dsDNA
• It pulls the 2 strands apart

Recruited by dnaA
Loaded around ssDNA
ATPase-dependent translocation
Strand exclusion model

Question 24

Stabalising ssDNA

Answer 24

Single stranded binding (SSB) protein
• Tetrameric protein
• ssDNA is wrapped around SSB tetramers
• Prevents secondary structure formation

Question 25

Prepriming complex

Answer 25

DnaA recruits dnaB | DnaB begins to translocate 5’ --> 3’

Question 26

Relaxing DNA molecules

Answer 26

``` Topoisomerase I Catalyses relaxation of supercoiled DNA 1) Cleavage of 1 strand 2) Passage of cut end under other strand 3) Resealing the break ```

Question 27

Untangling DNA molecules

Answer 27

``` Topoisomerase II Catalyses untangling of DNA duplexes 1) Cleavage of both strands 2) Passage separate duplex molecule through break 3) Resealing the break ```

Question 28

Primase

Answer 28

``` RNA primer – not DNA primer • RNA was formed earlier = ancestral artefact Required for the synthesis of DNA Provides initial free 3’ OH Primase can initiate but not extend ``` DnaG primase • Synthesises RNA primer • Recruited by dnaB

Question 29

DNA polymerase III

Answer 29

``` Involved in DNA replication DNA polymerase core made up of: 1) Polymerase unit - alpha 2) Exonuclease domain - e 3) Sliding clamp - B2 ```

Question 30

What does the polymerase unit (alpha) do?

Answer 30

Has fingers, palm & thumb 5' to 3' DNA synthesis Geometry enforces Watson-Crick base pairing Adds one base onto the DNA strand

Question 31

What does the exonuclease domain (e) do?

Answer 31

3' to 5' exonuclease | Proofreading

Question 32

What does the sliding clamp (B2) do?

Answer 32

``` Beta subunit is a ring Hole big enough for dsDNA Allows polymerase III to stay in contact with the DNA strand at all times • Alpha subunit can be more efficient • Can produce lots of DNA quickly • Doesn't rely on diffusion events ```

Question 33

The lagging strand problem

Answer 33

DNA polymerase synthesises 5' to 3' Lagging strand grows 3' to 5' Forms Okazaki fragments Lagging strand loops back on itself so DNA polymerase can still move in the same direction

Question 34

DNA polymerase III holoenzyme

Answer 34

DnaB recruits 2 DNA polymerases for coordinated synthesis of the leading and the lagging strand

Question 35

The trombone model of the lagging strand

Answer 35

Looping of lagging strand Lets go after adding 1000nts New loop then formed Loop lengthens & shortens like a trombone slide Loop is short at the start of the Okazaki fragment When finishing the fragment the loop is a lot bigger

Question 36

Filling in the gaps in the lagging strand

Answer 36

``` DNA polymerase I • Fills in gaps between fragments • 5’ to 3’ exonuclease activity • Removes RNA primer & synthesises DNA • Slow (10nt/s) ``` DNA ligase • Catalyses phosphodiester bond formation between 3’ hydroxyl and 5’ phosphate • Fills in ‘nicks’ between Okazaki fragments