Brainscape's Knowledge GenomeTM

Browse over 1 million classes created by top students, professors, publishers, and experts.


See full index

biochemistry of nucleic acids > helicases > Flashcards

helicases Flashcards

1
Q

what is a helicase?

A

an enzyme which is able to convert free energy from hydrolysis of NTP into unwinding of nucleic acid duplexes

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

what are helicases used for?

A

any process which requires the thermodynamically unfavourable separation of base pairs to access ssDNA

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

give examples of the type of processes helicases are used in

A
  • replication
  • repair
  • recombination
  • transcription
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

what are the requirements of a helicase?

A
  • must be able to bind nucleic acid
  • must bind and hydrolyse NTP
  • must carryout hydrolysis dependent unwinding
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

what is meant by ‘unwinding’?

A
  • moving along nucleic acid (translocation)
  • separating strands (duplex destabilisation)
  • clearing the path (snowploughing)
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

by what factors are helicases classified?

A
  • direction of movement
  • structural features
  • template affected
  • number of subunits
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

briefly outline how to conduct an unwinding assay

A
  • 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 well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

how can you identify if helicase activity is present from an unwinding assay?

A

is there is no helicase activity, the gel will run slowly as large circular DNA is still bound to a small labelled section

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

what does an unwinding assay tell us? what does it not tell us?

A

shows if helicase activity is present, but does not show direction of movement

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

give an example of a helicase family which is defined structurally as a helicase but has no unwinding activity

A

Swi2/Snf2 has no unwinding activity but are involved in chromatin remodelling

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

what are the features of the N23 helicase family?

A

3’-5’ RNA helicase from hepatitis C that can use any NTP or dNTP

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

what are the features of the eIF4a helicase family?

A

eukaryotic RNA helicase, reversible and uses ATP or dATP

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

what is the active rolling model?

A

helicases must have two or more subunits, which bind in turn to dsDNA, separating the strands and remaining anchored to ssDNA before rolling over

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

what are the problems with the active rolling model?

A
  • many helicases are active as monomers

- step size problem - step size is smaller than binding, helicase would have to slip backwards

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

what is meant by the inchworm model?

A

helicase slides along one strand in an alternating process of two contacts and a single contact which permits sliding

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

what is PcrA?

A

the first crystal structure for a helicase, isolated from bacillus stearothermophilus. this enzyme is involved in DNA repair and rolling circle replication

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
17
Q

how was ADPNP used to investigate PcrA?

A

ADPNP is a non-hydrolysable substrate analogue for ATP

allows comparison of ATP bound and non-ATP bound forms

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
18
Q

what is the mechanism of PcrA?

A

the signature motifs are between 1a and 2a domains, the ATP forms a cross link, joining the two domains together, permitting translocation

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
19
Q

how does PcrA move?

A

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

20
Q

how can direction of helicase movement be determined experimentally?

A
  • 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
21
Q

what is the functional purpose of many aromatic residues in the helicase domain of PcrA?

A

these form a series of stacking pockets ‘an ssDNA processing conveyor belt’

22
Q

how does the binding of ATP to the helicase domain of PcrA facilitate translocation?

A

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

23
Q

how do helicases stop ssDNA strands from re-annealing?

A

the shape of the protein is complementary to the DNA, also, the separated strands go down different ‘channels’ in the protein

24
Q

in crystallographic analysis of helicases, what has been found in all 4 monomeric helicases?

A

in all, hydrolysis of 1ATP achieves a net movement of one base

25
Q

what does the finding that 1ATP -> NET 1 base movement indicate?

A

the active rolling model is incorrect

26
Q

what is snowploughing?

A

the idea that the extra energy not used for separation of bases is used to displace other bound proteins such as transcription factors

27
Q

how did experiments using Dda and gp41 provide evidence for snowploughing?

A

on an artificial dsDNA template, helicase had the ability to push streptavidin off biotin (an extremely tight interaction)

28
Q

in the experiments using streptavidin and biotin, why was there an excess of free biotin in solution?

A

to trap displaced biotin, preventing it from re-attaching

29
Q

in the experiments providing evidence for snowploughing, how did they rule out spontaneous dissociation?

A

performed the experiment where there was:

  • no helicase or ATP
  • helicase but no ATP
  • helicase and ATP
30
Q

what was found when they added extra helicases in the streptavidin experiments?

A

extra helicases displace biotin faster, but to a limit

31
Q

what is RecBCD?

A

a heterotrimeric helicase - catalyses a complex reaction in which double stranded DNA breaks are processed prior to repair by homologous recombination. RecB and RecD have helicase activity

32
Q

what are the characteristics of RecB?

A

3’-5’ helicase
from SF1 family
each domain is similar to those in PcrA but the orientation of 1B and 2B is very different

33
Q

what are the characteristics of RecD?

A

similar 1a, 2a architecture

5’-3’ helicase of the SF1 family

34
Q

how can we show that both helicases in RecBCD are functional?

A

using site-directed mutagenesis to make changes to the ATP binding domain to render it non-functional
RecBCD - functional
RecBCD - functional
RecBCD - non-functional

35
Q

what are the characteristics of RecC?

A

helicase-like, but no unwinding activity

contains a ‘pin’ which helps direct the strands down different channels

36
Q

what are the suggested advantages of having 2 helicases in complex?

A

processivity - how far along the enzyme moves before it falls off
speed
step - the ability of the complex to step over a DNA break

37
Q

what process are hexameric helicases commonly associated with?

A

DNA replication forks

38
Q

give examples of hexameric 5’-3’ helicases

A

DnaB in e.coli

bacteriophage t7 gp4

39
Q

give examples of hexameric 3’-5’ helicases

A

eukaryotic MCM2-7, papillomavirus E1

40
Q

what is the structure of bacteriophage T7 gp4 helicase?

A

predominantly negative charge around outside, positive charge on the inside, facilitates interaction of ssDNA/RNA on inside

41
Q

why is it difficult to get crystallographic images of helicases?

A

requires a protein which normally moving to be static

42
Q

what are the models for hexameric helicase activity?

A

wedge
torsional
helix destabilising

43
Q

what are the features of the the ‘wedge’ model?

A

there is specific interaction with only the central, included strand.
the excluded strand is displaced in a non-specific way
there is no contact with the duplex region

44
Q

what are the features of the torsional model?

A

the helicase interacts with both the included and excluded strands. the excluded strands act as a fulcrum promoting rotation. there is no contact with the duplex region, but torque generated by rotation destabilises the duplex

45
Q

what are the features of the helix-destabilising model?

A

surface of the helicase makes direct contacts with the duplex region

biochemistry of nucleic acids (19 decks)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2024 Bold Learning Solutions. Terms and Conditions