helicases Flashcards
what is a helicase?
an enzyme which is able to convert free energy from hydrolysis of NTP into unwinding of nucleic acid duplexes
what are helicases used for?
any process which requires the thermodynamically unfavourable separation of base pairs to access ssDNA
give examples of the type of processes helicases are used in
- replication
- repair
- recombination
- transcription
what are the requirements of a helicase?
- must be able to bind nucleic acid
- must bind and hydrolyse NTP
- must carryout hydrolysis dependent unwinding
what is meant by ‘unwinding’?
- moving along nucleic acid (translocation)
- separating strands (duplex destabilisation)
- clearing the path (snowploughing)
by what factors are helicases classified?
- direction of movement
- structural features
- template affected
- number of subunits
briefly outline how to conduct an unwinding assay
- provide helicase, ATP and magnesium to a short section of DDNA hybridised to circular DNA that has been labelled
- run gel under non-denaturing conditions
- use negative and positive control
how can you identify if helicase activity is present from an unwinding assay?
is there is no helicase activity, the gel will run slowly as large circular DNA is still bound to a small labelled section
what does an unwinding assay tell us? what does it not tell us?
shows if helicase activity is present, but does not show direction of movement
give an example of a helicase family which is defined structurally as a helicase but has no unwinding activity
Swi2/Snf2 has no unwinding activity but are involved in chromatin remodelling
what are the features of the N23 helicase family?
3’-5’ RNA helicase from hepatitis C that can use any NTP or dNTP
what are the features of the eIF4a helicase family?
eukaryotic RNA helicase, reversible and uses ATP or dATP
what is the active rolling model?
helicases must have two or more subunits, which bind in turn to dsDNA, separating the strands and remaining anchored to ssDNA before rolling over
what are the problems with the active rolling model?
- many helicases are active as monomers
- step size problem - step size is smaller than binding, helicase would have to slip backwards
what is meant by the inchworm model?
helicase slides along one strand in an alternating process of two contacts and a single contact which permits sliding
what is PcrA?
the first crystal structure for a helicase, isolated from bacillus stearothermophilus. this enzyme is involved in DNA repair and rolling circle replication
how was ADPNP used to investigate PcrA?
ADPNP is a non-hydrolysable substrate analogue for ATP
allows comparison of ATP bound and non-ATP bound forms
what is the mechanism of PcrA?
the signature motifs are between 1a and 2a domains, the ATP forms a cross link, joining the two domains together, permitting translocation
how does PcrA move?
the two domains move relative to each other
a - ssDNA held by 1a
b- ATP binds between 1a and 2a, acting as a cross bridge, causing DNA binding
c-atp hydrolysis reverses movement
how can direction of helicase movement be determined experimentally?
- set up a partial duplex template
- cleave using an enzyme that cut off centre
- examine the size of the labelled strand displaced by the helicase
what is the functional purpose of many aromatic residues in the helicase domain of PcrA?
these form a series of stacking pockets ‘an ssDNA processing conveyor belt’
how does the binding of ATP to the helicase domain of PcrA facilitate translocation?
upon binding of ATP, phe626 forms an interaction with base 6, phe64 pushes out an interaction with base two and the amino acid adjacent to it (tyrosine) when ATP is hydrolysed, phe626 swings out of the way. as phe64 moves away to permit base 3 to form interaction to tyrosine. the effect of this is the bases shuffle forward
how do helicases stop ssDNA strands from re-annealing?
the shape of the protein is complementary to the DNA, also, the separated strands go down different ‘channels’ in the protein
in crystallographic analysis of helicases, what has been found in all 4 monomeric helicases?
in all, hydrolysis of 1ATP achieves a net movement of one base
