Flashcards in Motors that change DNA: helicases Deck (50):
What is the importance of DNA?
The accurate and faithful replication and translation of DNA is essential
Multiple proteins are involved in ensuring the fidelity of these processes
"Elegant enzymatic choreography" Nelson and Cox
E.g. DNA replication
When were helicases first identified?
In the 1970s
What do helicases have the ability to do?
Convert free energy released by hydrolysis of NTP (usually ATP) into the unwinding of the nucleic acid duplex (DNA:DNA, RNA:RNA, or DNA:RNA)
In what organisms are helicases found?
They are ubiquitous, i.e. found in viruses, bacteria and eukaryotes
Give an example of why helicases are divergent
E.g. E. coli: at least 14 different helicases
Helicases are essential in all processes that require the thermodynamically unfavourable separation of base pairs to access single-stranded DNA (ssDNA) such as..
(Errors in any of theses processes lead to diseases)
Give examples of diseases that are caused by a helicase mutation
What must a helicase be able to DO in order to carry out unwinding reactions?
Involves at least 3 components:
-Bind nucleic acid
-Bind and hydrolyse NTP
Unwinding now recognised to involve at least 2 processes:
-Moving along the nucleic acid (translocation)
-Separating strands (duplex destabilisation)
List the several ways that helicases can be classified
Direction of movement: 3' to 5' vs. 5' to 3' (note: can achieve same NET movement depending upon which strand is template)
Structural features: 4+ superfamilies recognised
Template affected: DNA vs. RNA
Number of subunits: hexamer vs. monomer (or dimer)
How can you determine the direction of movement of a helicase?
1. Set up partial duplex template
2. Cleave using enzyme that cuts off-centre
3. Examine size of labelled strand displaced by helicase
Helicases are divided into 4 (or more) superfamilies based on the possession of what?
"Helicase signature motifs"
E.g. superfamily 2 (SF2) - 7 motifs, family includes:
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
i.e. variable proerties within class
What do members of a structural superfamily not necessarily share?
Other preferences (NTP usage, direction, template specificity)
Some enzymes defined structurally as 'helicases' have an effect on DNA but do not appear to have any what?
Unwinding activity - better 'translocase motifs'?
E.g. Swi2/Snf2 helicase family (also in SF2) have no unwinding activity, but are involved in chromatin remodelling
What are the two principal models that have been considered for helicase movement along a template?
"Active rolling" model
Describe the "active rolling" model
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
Describe the "inchworm" model
Helicase slides along one strand
Alternates between 1 and 2 contact points on strand to achieve net movement
What is the structural problem with the "active rolling" model?
The structure of several helicases are now solved and many are definitely active as monomers (active rolling requires at least dimers)
What is the step size problem with the "active rolling" model?
'Footprint experiments show that various helicases 'protect' 8-10 bases of ssDNA, but work looking at the helicase 'step size' have calculated 1 bp up to a maximum of 4-5 bp - to reconcile these would require the helicase to 'slip' backwards
What is the approved model by which at least some helicases move along a template?
The "inchworm" model
Are the structural and step size problems a problem for the "inchworm" model?
Name and describe the first crystal structure for any helicase by Subramanya et al., in 1996
PcrA from Bacillus stearothermophilus
PcrA is an essential enzyme, involved in both DNA repair and rolling-circle replication
Is PcrA a monomer or dimer?
What direction does the PcrA helicase work in?
The 3' to 5' direction
What superfamily group is the PcrA helicase a member of?
The SF1 group
What is ADPNP?
A "non-hydrolysable ATP analog"
How does the helicase stop ssDNA strands from rejoining?
There is a complementary shape of protein surface
ssDNA is physically separated so that the strands cannot reanneal
What did Von Hippel and Delagoutte hypothesise in 2001 about ATP usage and movement? And explain is the reality?
Thermodynamically, the hydrolysis of 1 ATP should provide sufficient energy to separate 6-8 bp of DNA
If, in reality, helicase only achieves 1 bp separation per ATP is this inefficiency?
Not necessarily! Helicase movement may sometimes require displacement of other bound proteins (e.g. transcription factors), the 'excess' energy may be needed to achieve this - "snowploughing"
Outline the evidence for "snowploughing" as carried out by Kevin Raney and his colleagues
Dda and gp41 are 5' to 3' helicases from bacteriophage T4
Dda functions as a monomer, gp41 as a hexamer
Set up experiments with biotin covalently attached to 3' end of dsDNA
Streptavidin binds to biotin - can helicase knock it off?
Set up experiment: need 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
Either helicase in the presence, but not the absence, of ATP displaced streptavidin faster than dissociation (gp41 = over 500 times faster, Dda = over million-fold)
Further experiment by same team highlights effect of additional helicase molecules
If template only long enough to bond one helicase = 'slow' (relative to later experiments)
Longer template, more helicase bound = faster
Catalyses complex reaction in which double-strand DNA breaks processed prior to repair by homologous recombination
RecBCD of interest as it involves TWO helicases
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
5' to 3' helicase
SF1 family, similar Dda
Domains 2 and 3 are equivalent to 1A and 2A
Domain 1 different
Do RecB and RecD have the same or different overall movement?
Although RecB and RecD have opposite orientations they have same overall movement as travel of different strands
Are both helicases functional in RecBCD?
How can you tell that both helicases in RecBCD are functional?
Use site-directed mutagenesis to make change in crucial ATP-binding domain (actually lysine to glutamine in each case), no longer efficient ATP hydrolysis
Call mutants RecB* and RecD*
- RecB*CD = functional helicase
- RecBCD* = functional helicase
- RecB*CD* double mutant = inactive
Does RecC contact one or both strands of DNA?
RecC contacts both strands of DNA and splits them at prominent 'pin'
RecB and RecD move along ssDNA pulling..?
dsDNA onto pin and aiding unwinding
Why have 2 helicases in complex?
Both helicases RecB and RecD use 1 ATP per base moved forward, i.e. twice as expensive, why bother?
Processivity? May boost processivity (i.e. how far complex travels before falling off)
- RecBCD = 30000 bp per binding event
Speed? May enhance speed of travel
- RecBCD = 1000 bp per second
Step? May allow complex to "step over" a ssDNA break
- Speculation at present
What sort of helicase structures are most prominently associated with DNA replication forks? And give examples
Tend to be hexameric ring structures
5' to 3'
E. coli DnaB
Bacteriophage T7 gp4
3' to 5'
Eukaryotic MCM2-7 (mini-chromosome maintenance)
Shown by EM and increasingly by crystallography
Describe the key features of the wedge hexameric helicase model
Specific interaction only with central ('included') strand
Excluded strand displaced in non-specific way
No contact with duplex region
Describe the key features of the torsional hexameric helicase 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
Describe the key features of the helix-destabilising hexameric helicase model
Surface of helicase makes direct contact with duplex region
Evidence from monomers suggests this may be 'real' model, but currently open to debate
Describe the features of the T7 gp4 helicase
DNA-binding loops of gene 4 protein located within central cavity of hexamer
Hexamer is asymmetric - there are 3 distinct monomers
4 out of 6 units have nucleotide bound
Describe the features of the E1 helicase
Crystal formed in presence of ssDNA, ADP and Mg2+
As with T7 gp4, the hexamer has:
- ssDNA in centre
- hairpins bind DNA (13An at narrowest)
- 3 subunit states:
Describe the E1 helicase mechanism
ssDNA nucleotides align with one nucleotide per subunit
Protein hairpins form a 'spiral staircase' that tracks ssDNA backbone
1 ATP hydrolysed per nucleotide translocated, i.e. 6 ATP per full cycle, with movement of 6 nucleotides
Unlike T7 gp4 which is considered to have a "bucket brigade" model, E1 is thought to have an..
"Escorted" system like cargo in wagons - contact maintained with same subunit throughout cycle
DNA replication is a highly..
DNA helicase are key enzymes in..
Replication and other DNA-associated processes
PcrA helicase is an example of a..
Monomeric helicase that works via an inchworm model
RecBCD is a..
Trimeric helicase with two helicase proteins and greater processivity