Motors that change DNA: helicases

Question 1

What is the importance of DNA?

Answer 1

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

Question 2

When were helicases first identified?

Answer 2

In the 1970s

Question 3

What do helicases have the ability to do?

Answer 3

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)

Question 4

In what organisms are helicases found?

Answer 4

They are ubiquitous, i.e. found in viruses, bacteria and eukaryotes

Question 5

Give an example of why helicases are divergent

Answer 5

E.g. E. coli: at least 14 different helicases

Question 6

Helicases are essential in all processes that require the thermodynamically unfavourable separation of base pairs to access single-stranded DNA (ssDNA) such as..

Answer 6

Replication
Repair
Recombination
Transcription
(Errors in any of theses processes lead to diseases)

Question 7

Give examples of diseases that are caused by a helicase mutation

Answer 7

Xeroderma pigmentosa
Werner's syndrome
Fanconi anaemia
Bloom syndrome
Cockayne syndrome
Rothmund-Thomson Syndrome

Question 8

What must a helicase be able to DO in order to carry out unwinding reactions?

Answer 8

Involves at least 3 components:
-Bind nucleic acid
-Bind and hydrolyse NTP
-Hydrolysis-dependent unwinding
Unwinding now recognised to involve at least 2 processes:
-Moving along the nucleic acid (translocation)
-Separating strands (duplex destabilisation)

Question 9

List the several ways that helicases can be classified

Answer 9

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)

Question 10

How can you determine the direction of movement of a helicase?

Answer 10

1. Set up partial duplex template
2. Cleave using enzyme that cuts off-centre
3. Examine size of labelled strand displaced by helicase

Question 11

Helicases are divided into 4 (or more) superfamilies based on the possession of what?

Answer 11

“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

Question 12

What do members of a structural superfamily not necessarily share?

Answer 12

Other preferences (NTP usage, direction, template specificity)

Question 13

Some enzymes defined structurally as ‘helicases’ have an effect on DNA but do not appear to have any what?

Answer 13

Unwinding activity - better ‘translocase motifs’?

E.g. Swi2/Snf2 helicase family (also in SF2) have no unwinding activity, but are involved in chromatin remodelling

Question 14

What are the two principal models that have been considered for helicase movement along a template?

Answer 14

“Active rolling” model

“Inchworm” model

Question 15

Describe the “active rolling” model

Answer 15

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

Question 16

Describe the “inchworm” model

Answer 16

Helicase slides along one strand

Alternates between 1 and 2 contact points on strand to achieve net movement

Question 17

What is the structural problem with the “active rolling” model?

Answer 17

The structure of several helicases are now solved and many are definitely active as monomers (active rolling requires at least dimers)

Question 18

What is the step size problem with the “active rolling” model?

Answer 18

‘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

Question 19

What is the approved model by which at least some helicases move along a template?

Answer 19

The “inchworm” model

Question 20

Are the structural and step size problems a problem for the “inchworm” model?

Answer 20

No, neither

Question 21

Name and describe the first crystal structure for any helicase by Subramanya et al., in 1996

Answer 21

PcrA from Bacillus stearothermophilus

PcrA is an essential enzyme, involved in both DNA repair and rolling-circle replication

Question 22

Is PcrA a monomer or dimer?

Answer 22

A monomer

Question 23

What direction does the PcrA helicase work in?

Answer 23

The 3’ to 5’ direction

Question 24

What superfamily group is the PcrA helicase a member of?

Answer 24

The SF1 group

Question 25

What is ADPNP?

Answer 25

A "non-hydrolysable ATP analog"

Question 26

How does the helicase stop ssDNA strands from rejoining?

Answer 26

There is a complementary shape of protein surface | ssDNA is physically separated so that the strands cannot reanneal

Question 27

What did Von Hippel and Delagoutte hypothesise in 2001 about ATP usage and movement? And explain is the reality?

Answer 27

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"

Question 28

Outline the evidence for "snowploughing" as carried out by Kevin Raney and his colleagues

Answer 28

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

Question 29

Describe RecBCD

Answer 29

Heterotrimeric helicase/nuclease Catalyses complex reaction in which double-strand DNA breaks processed prior to repair by homologous recombination RecBCD of interest as it involves TWO helicases

Question 30

Describe RecB

Answer 30

``` 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 ```

Question 31

Describe RecD

Answer 31

5' to 3' helicase SF1 family, similar Dda Domains 2 and 3 are equivalent to 1A and 2A Domain 1 different

Question 32

Do RecB and RecD have the same or different overall movement?

Answer 32

Although RecB and RecD have opposite orientations they have same overall movement as travel of different strands

Question 33

Are both helicases functional in RecBCD?

Answer 33

Yes

Question 34

How can you tell that both helicases in RecBCD are functional?

Answer 34

``` 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 ```

Question 35

Does RecC contact one or both strands of DNA?

Answer 35

RecC contacts both strands of DNA and splits them at prominent 'pin'

Question 36

RecB and RecD move along ssDNA pulling..?

Answer 36

dsDNA onto pin and aiding unwinding

Question 37

Why have 2 helicases in complex?

Answer 37

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

Question 38

What sort of helicase structures are most prominently associated with DNA replication forks? And give examples

Answer 38

``` Tend to be hexameric ring structures - E.g: 5' to 3' E. coli DnaB Bacteriophage T7 gp4 - E.g: 3' to 5' Eukaryotic MCM2-7 (mini-chromosome maintenance) Papillomavirus E1 Shown by EM and increasingly by crystallography ```

Question 39

Describe the key features of the wedge hexameric helicase model

Answer 39

Specific interaction only with central ('included') strand Excluded strand displaced in non-specific way No contact with duplex region

Question 40

Describe the key features of the torsional hexameric helicase model

Answer 40

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

Question 41

Describe the key features of the helix-destabilising hexameric helicase model

Answer 41

Surface of helicase makes direct contact with duplex region | Evidence from monomers suggests this may be 'real' model, but currently open to debate

Question 42

Describe the features of the T7 gp4 helicase

Answer 42

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 Proposed mechanism

Question 43

Describe the features of the E1 helicase

Answer 43

``` Crystal formed in presence of ssDNA, ADP and Mg2+ As with T7 gp4, the hexamer has: - asymmetry - ssDNA in centre - hairpins bind DNA (13An at narrowest) - 3 subunit states: ATP-bound ADP-bound Empty (Apo) ```

Question 44

Describe the E1 helicase mechanism

Answer 44

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

Question 45

Unlike T7 gp4 which is considered to have a "bucket brigade" model, E1 is thought to have an..

Answer 45

"Escorted" system like cargo in wagons - contact maintained with same subunit throughout cycle

Question 46

DNA replication is a highly..

Answer 46

Complex process

Question 47

DNA helicase are key enzymes in..

Answer 47

Replication and other DNA-associated processes

Question 48

PcrA helicase is an example of a..

Answer 48

Monomeric helicase that works via an inchworm model

Question 49

RecBCD is a..

Answer 49

Trimeric helicase with two helicase proteins and greater processivity

Question 50

T7 gene 4 and E1 helicases are..

Answer 50

Hexameric rings that translocate ssDNA through a central hole