helicases Flashcards
helicases =
- are able to convert free energy released by hydrolysis of nucleoside triphosphates (usually ATP) inorder to unwind nucleic acid duplexes (DNA:DNA, RNA:RNA or DNA:RNA)
- are ubiquitous (found in all organisms)
- are divergent (e.g. E.coli has 14 different helicases)
- are essential in all processes that require the thermodynamically unfavourable separation of base pairs to access ssDNA (replication, repair, recombination, transcription)
helicase mutations lead to…
diseases such as…
- xeroderma pigmentosa (hypersensitivity to light)
- bloom syndrome
- fanconi anaemia
requirements of helicases:
- bind nucleic acid
- bind and hydrolyse nucleoside triphosphates
- perform hydrolysis-dependent unwinding
unwinding may include these processes…
- moving along the nucleic acid (translocation)
- separating strands (duplex destabilisation)
- clearing the path ( snowploughing)
ways helicases can be classified:
- the direction of movement (3’ to 5’ OR 5’ to 3’)
- structural features (6+ superfamilies recognised)
- template effected (DNA or RNA)
- number of subunits (hexamer, monomer or dimer)
helicase direction of movement:
translocates along template strand and causes separation of the strands either…
from 3’ to 5’ in superfamilies 1A and 2A helicases
OR
from 5’ to 3’ in superfamilies 1B and 2B helicases
determining whether something is a helicase:
unwinding assay =
- a labelled short section of DNA is hybridised onto a circle of ssDNA
- when a gel is run you can identify if the helicase has worked by seeing if the labelled shorter section has separated from the circle
- if no helicase is present = unseparated larger complex doesn’t run as fast on gel
- if heated as a control = separates strands = band runs further on gel
- if helicase is present = runs faster due to separation
determining the direction of movement of a helicase:
- attach a short section of DNA to a circle of ssDNA forming a partial duplex template
- cleave using a restriction enzyme
- label ends of strands
- carry out a helicase reaction to unwind
- run on a non-denaturing gel and analyze
- examine the size of labelled strand displaced by helicase
helicase structural features:
- helicases are divided into 6 (or more) superfamilies based on possession of ‘helicase signature motifs’
- members of a structural superfamily do not necessarily share other preferences:
e. g. may have different NTP usage, direction of movement or template specificity
some enzymes defined structurally as helicases have no unwinding activity…
e.g. Swi2/Snf2 helicase family have no unwinding activity but have an effect on DNA as they are involved in chromatin remodelling
two models have been proposed for how helicases move along the template strand…
active rolling model + inchworm model
active rolling model =
helicase must have 2 or more subunits which bind in turn to dsDNA, separate strands and remain anchored to ssDNA before rolling so that the other unit takes over
problems with the active rolling model…
- structural studies show that many helicases are active a monomers (active rolling requires at least a dimer)
- step size issue: this method would require the helicase to slip backwards (which would be unlikely)
- these are not problems with the inchworm model
inchworm model =
helicase slides along one strand, alternating between two contact points and a single contact point on the DNA strand to achieve net movement
- this has been shown in the helicase PcrA
helicase PcrA mechanism:
there are four separate domains: 1A, 2A, 1B, 2B
- ssDNA held by 1A - 1A is bound strongly and 2A is bound loosely
- ATP binds in the cleft between 1A and 2A and acts as a cross-bridge causing 1B and 2B to bind to dsDNA
= ATP binding allows movement resulting in a change in strength for the domains = alternates which domain is bound strongly
- ATP hydrolysis reverses movement
