Helicases and Topoisomerases Flashcards
(83 cards)
1
Q
What are helicases?
A
Identified in 1970’s
Ability to convert free energy released by hydroloysis of NTP (usually ATP) into unwinding of nucleic acid duplex (DNA:RNA), (RNA:RNA) OR (DNA:RNA)
Ubiquitous (found in viruses, bacteria, and eukaryotes)
Divergent (E. coli has 14 different helicases)
2
Q
What are helicases essential for?
A
Ability to access single stranded DNA
In all processes that require thermodynamically unfavourable separation of base pairs to single-stranded DNA (ssDNA)
3
Q
What are the 4 processes for helicases?
A
- Replication
- Repair
- Recombination
- Transcription
Errors in any of these processes lead to diseases
4
Q
What are some diseases that can be caused through helicase mutation?
A
- Xeroderma pigmentosa
- Fanconi anaemia
- Bloom syndrome
- Rothmund-Thomson Syndrome
- Werners syndrome
- Cockayne syndrome
5
Q
What 3 things must a helicase be able to do in order to carry out unwinding reactions?
A
- bind nucleic acid
- bind and hydrolyse NTP
- hydrolysis-dependent unwinding (or other)
6
Q
How many processes are involved to recognise unwinding?
A
3
- Moving along the nucleic acid (TRANSLATION)
- Separating strands (DUPLEX DESTABILISATION)
- Clearing the path (SNOWPLOUGHING)
7
Q
Which helicase family have no unwinding activity?
A
Swi2/Snf2 helicase family
But are involved in chromatin remodelling
8
Q
What are the several ways in which a helicase can be classified?
A
- Direction of movement
Are they going from 3’ to 5’ or 5’ to 3’ - Structural features
6+ Superfamilies recognised - Template affected
DNA vs RNA - Number of subunits
Hexamer vs Monomer (dimer)
9
Q
What is the direction of movement in helicases?
A
DNA template is read from 3’ to 5’ but new strand is synthesised in the 5’ to 3’ direction
Can achieve same NET movement depending upon which strand is template
10
Q
What is DNA unwinding assay?
A
Designed to investigate whether a compound gets inserted into the DNA double helix OR binds in the groove
11
Q
How do we determine the direction of movement in unwinding assay?
A
• Set up partial duplex template • Cleave using enzyme that cuts off-centre • Examine size of labelled strand displaced by helicase
12
Q
What are the structrual features of helicases?
A
- Helicases are divided into 6+ superfamilies
- Members of the same family do not share other preferences
- Some enzymes have an effect on DNA but do not appear to have any unwinding activity
13
Q
What motifs does Superfamily 2 (SF2) include?
A
• NS3 3’ to 5’ RNA helicase from Hepatitis C can use any NTP or dNTP
• eIF4a eukaryotic RNA helicase, reversible
can use only ATP or dATP
• UvrB involved in DNA repair in prokaryotes
• RecG rescues stalled replication forks
14
Q
What are the 2 models of movement along the template strand?
A
- Active rolling model
2. Inchworm model
15
Q
What is active rolling model?
A
- Helicase must have 2 or more subunits
- Bind in turn to dsDNA separate strands and remain anchored to ssDNA before rolling so that other unit takes over
16
Q
What is the inchworm model?
A
- Helicases slides along one strand
- Alternates between 1 and 2 contact points on strand to achieve net movement
17
Q
What are the problems with active rolling model?
A
- Structure of several helicases now solved
- Step size: new experiements show that helicases protect 8-10 bases of ssDNA but work looking at the helicase “step size” have calculated 1 bp up to maximum of 4-5 bp
18
Q
What is PcrA?
A
An enzyme involved in both DNA repair and rolling circle replication
Monomer
SF1 member
3’ to 5’ direction
19
Q
What is PcrA’s interaction with dsDNA?
A
- ssDNA gets held by motif 1A
- ATP binds in cleft between 1A and 2A and acts as a cross-bridge to bring them closer together
- The resulting action of ATP binding is causing pulling of 2B and 1B to bind dsDNA
- ATP hydrolysis reverses movement
20
Q
What is ADPNP?
A
- Non-hydrolysable ATP analog
- Looks like ATP
21
Q
What is the difference between ADPNP and ATP?
A
Phosphate bond contains a nitrogen instead of oxygen
22
Q
What is the importance of ADPNP?
A
If we want to identify structure and how structure fits with mechanism, we want two different versions
One with ATP bound
One without ATP bound (ADPNP) to trap it
Compare the two structures and analyse them
23
Q
Where are the signature motifs based?
A
Between the 1A and 2A
They are crucial for binding to ATP
24
Q
What does the benzene ring allow for?
A
For ring stacking between the base and the amino acid
NOT TRUE FOR HISTADINE AND ARGININE
25
Where does the F626 amino acid come from?
The 2A domain
26
Where does the F64 amino acid come from?
The 1A domain
27
What is the ssDNA processing conveyvor belt?
Aromatic residues form series of stacking pockets
28
What happens when ATP becomes binded?
F626 (RED) creates a new binding pocket for base 6
29
What does F64 (GREEN) do?
It occupies the pocket formed by Y257 (BLUE), forcing base 2 to move into position previously occupied by base 1
30
What happens when ATP is hydrolysed?
F626 moves away again, releasing grip on base 6
F64 moves away again, allowing base 3 to move into Y257 pocket
Bases 4-6 shuffle into next postition
Net movement: 1 bp along per ATP hydrolysed
31
How do we stop ssDNA strands from re-joining?
There is a complementary shape of protein surface
ssDNA physically separated, strands cannot re-anneal due to physical arrangement
32
What are the 4 different proteins of which there is a connection between ATP and translocation?
- PcrA (SF1A)
- NS3 (SF2A)
- RecD2 (SF1B)
- DinG (SF2B)
Each has a different characteristic
33
What does all 4 proteins share?
That hydrolysis of 1 ATP achieves net movement of 1 base
34
Thermodynamically, how many base pairs should we move forward through hydrolysis of ATP?
We should provide sufficient energy to separate 6-8 bp of DNA
35
What might helicase movement sometimes require?
Displacement of other bound proteins (transcription factors), the excess energy may be needed to achieve snowploughing
36
What is the evidence for snowploughing?
- Dda and hp41 are 5' to 3' from bacteriophage T4
- Dda functions as a monomer and gp41 as a hexamer
- At 3' end they attached biotin which contains streptavidin
37
How to see if helicase can knock off streptavidin?
Make an experiment to free excess free biotin to trap displaced streptavidin so that it cannot reattach
Need to rule out spontaneous dissociation and to see if effect is ATP-dependent, so carry out:
- No helicase, no ATP
- Helicase, no ATP
- Helicase with ATP
38
Which one of the three experiments would knock streptavidin off?
HELICASE WITH ATP
39
How do we make the experiment faster?
Additional helicase molecules
Longer template, more helicase bound = faster
40
What is RecBCD?
- Heterotrimeric helicase/nuclease
| - It catalyses complex reaction in which double strand DNA breaks processed prior to repair by homologous recombination
41
How many active helicases does RecBCD have?
2
RecB
RecD
42
What is RecB?
3' to 5' helicase and nuclease
SF1 family, similar structure to PcrA
Each domain (1A, 1B, 2A, 2B) similar to those in PcrA but orientation of 1B and 2B very different
Extra arm on 1B contacts DNA
Extra nuclease domain via linker
43
What is RecD?
5' to 3' helicase
SF1 family, similar to Dda
Domains 2 and 3 are equivalent to 1A and 2A
Domain 1 is different
44
Do both helicases work in RecBCD?
Yes
45
How could we tell if both helicases work in RecBCD?
By using site-directed mutagenesis to make a change in crucial ATP-binding domain (usally lysine to glutamine in each case), no longer efficient ATP hydrolysis
A mutated B and D means = inactive complex
46
What happens if both B and D are mutated?
Mutated B and D means = inactive complex so cannot translocate across the template
47
What is RecC?
Contains a 5' channel to RecD
RecB binding cavity
RecC contacts both strands of DNA and splits them at prominent pin
48
What do RecB and RecD do together?
RecB and RecD move along ssDNA pulling dsDNA onto pin and aid in unwinding
49
Why have 2 helicases in complex?
Proccesivity - How far complex travels before falling off
Speed - May enhance speed of travel
Step - May allow complex to 'step over' a ssDNA break
RecD moves faster than RecB
Causes a loop of ssDNA ahead of complex
Important in function of enzyme to repair dsDNA breaks
50
What are hexameric helicases?
Helicases most prominently associated with DNA replication forks tend to be hexameric ring structures
5' to 3' or 3' to 5'
51
What are some 5' to 3' hexameric helicases?
- E. coli DnaB
- bacteriophage T7 gp4
Moves on the leading strand
52
What are some 3' to 5' hexameric helicases?
- eukaryotic MCM2-7
- papillomavirus E1
Attached to the lagging strand
53
How are hexameric helicases shown?
By EM and increasingly by crystallography
54
Name 3 hexameric helicase models?
1. Wedge
2. Torsional (Strand exclusion with wrap)
3. Helix-destabilising model
55
What is a wedge model?
- Specific interaction only with central strand
- Excluded strand displcaced in non-specific way
- No contact with duplex region
56
What is the torsional model?
- Helicase interacts with both the included and excluded strands
- Excluded strand acts as fulcrum promoting rotation
- No direct contact with duplex region, but torque generated by rotation destabilises duplex
57
What is helix-destabilising model?
- Surface of helicases makes direct contact with duplex region
- Evidence from momomers suggests this may be 'real' model
58
What is the T7 gp4 helicase mechanism?
- DNA binding loops of gene 4 protein located within central cavity of hexamer
- Hexamer is asymmetric, there are 3 distinct monomers like they're going through a cycle
- 4 out of 6 units have nucleotide bound
59
What is the E1 helicase mechanism?
- Crystal formed in the presence of ssDNA, ADP and Mg2+
- The hexamer has;
- assymetry
- ssDNA in centre
- hairpins bind DNA (13A at narrowest)
- 3 subunit states:
- - ATP bound
- - ADP bound
- - Empty
- ssDNA nucleotides align with one nucleotide per subunit
- protein hairpins form spiral staircase that tracks ssDNA backbone
60
What are topoisomerases?
An example is DNA gyrase
They are enzymes that alter the topological state of DNA in cells
Interchange between relaxed and supercoiled DNA in bacteria
61
What is Topo IV?
They can take out supercoils
Another process it has is, decatination
It puts a double strand break in
First identified in mutants that couldn't decatinate sufficiently
62
What is decatenation?
Decatenation is defined as the process of separating this physical linkage
63
What can both DNA gyrase and topo IV do?
They can both relax a supercoil
64
What are negative supercoils?
Have different orientations to postive supercoils
Only DNA gyrase can introduce negative supercoils
65
What are type II topoisomerases?
DNA gyrase
Topo IV
Breaks both strands
66
What is the mechanism for which type II strands are broken?
It involves an active site tyrosine and a phosphate group on the DNA backbone
67
What are the properties of E. coli DNA gyrase?
A protein
- Gene = gyrA
- Mol weight = 90 kDA
- Major role = breakage and reunion
- Drug interactions = Quinolones
B protein
- Gene = gyrB
- Mol weight = 90 kDA
- Major role = ATPase reaction
- Drug interactions = Coumarins
68
What are the properties of E. coli Topo IV?
A protein
- Gene = parC
- Mol weight = 84 kDA
- Major role = breakage and reunion
- Drug interactions = Quinolones
B protein
- Gene = parE
- Mol weight = 70 kDA
- Major role = ATPase reaction
- Drug interactions = Coumarins
69
What is the difference between eukaryotes and prokaryotes in type II topoisomerases?
The gene products are linked in eukaryotes but in prokaryotes they are seperated
70
How do we study the domains in proteins?
In isolation (break into two)
GyrA
1. Break the DNA with reunion domain, interaction with quinolones
2. DNA wrapping
GyrB
1. Interaction with coumarins
2. Interaction with GyrA, DNA and quinolones
71
Where does tyrosine form a covalent link on DNA gyrase?
It forms a covalent link on the 5' end
Leaves a 3' end where it breaks
72
What forms after the DNA gyrase breaks twice from covalent links from tyrosine?
A gate forms to which a point where DNA is going to be passed
73
What is the DNA cleavage mechanism?
Contains 2 Mg2+ ions
One polarises the hydroxyl group to the extent where it makes it easier for a histadine to pinch a proton which means the oxygen is able to take out a nucleophillic attack on the phosphate on the DNA backbone
5' phosphate is partailly stablised thanks to the Mg2+ bonds also sit with some negativity charged aspartic groups
Acid ends up protonating the 3' end and 5' covalently attached to the gyrase
74
What is the Topo II cycle?
- Section of DNa is bound
- Subsequent binding of ATP closes top gate, trapping a second piece of DNA
- The G-segment is then cleaved, forming a gate. The ends of DNA are 'held' via covalent bond to Tyr
- This step MAY involve hydrolysis of one ATP
- A little extra finger comes out of the top of the GyrB which is crucial to the mechanisms
75
What are the differences between topos?
- The catalytic mechanism seems to be true for all Type II topos
- The specific ability of DNA gyrase to put supercoils into DNA must come from structural differences
- The major difference is the inclusion of an extra C-terminal domain (CTD) on bacteiral Type II topos, which eukaryotic DO NOT HAVE
Topo IV can only loosely wrap some DNA, the positive charge is important for holding that orientation
Topo IV cannot introduce supercoils
76
What is GryA CTD?
CTD is a disc formed from 6 blades
Has 4 beta strands in it
4 of the 6 blades have positively charged surface
Expectation that DNA is wrapped around outside
77
What is florescence resonance energy transfer (FRET)?
A template DNA molecule to which florescent DNA molecules are added to each side of the DNA
A donor chromophore, initially in its electronic excited state, may transfer energy to an acceptor chromophore through nonradiative dipole–dipole coupling
78
How do we make proteins duller?
We use alanine scanning
Gyrase mutants in which this sequence is deleted, or replaced by 7 alanines
79
Why is ATP required for Type II topos?
As negative supercoils is energy requiring
Topo IV and eukaryotic Topo II need ATP to carry out a similar reaction
80
What are the inhibitors of gyrase and topo IV?
Novobiocin is not structurally similar to ATP but binds at an overlapping site inhibiting supercoiling
81
How does novobiocin bind to ATP?
ADPNP and novobiocin bind at the same site
82
Why are coumarins not useful to humans?
Due to their toxicity but they point the the way to other potentially useful drugs
83
What are the best known inhibitors of bacterial type II topos?
Fluoroquinolines
