HELICASES
Batlle Masó, Laura
Rosich Sangrà, Elena
Sumarroca Bordas, Marina
Torrecilas Testa, Tatiana
INDEX
A.
B.
C.
D.
E.
F.
Introduction
Materials and methods
Monomeric helicase: PcrA
Hexameric helicase: DnaB
Alignments and Superimpositions
Conclusion
INTRODUCTION
1.
2.
3.
4.
5.
Definition and function
Superfamilies
Hexameric helicases
RecA like and AAA+ domains
Evolution
1. Definition and Function

Helicases are enzymes that unwind duplex DNA, RNA or
DNA-RNA hybrids. They use energy derived from ATP
hydrolisis to separate base-paired nucleic acids.
They play roles in cellular processes which involve nucleic acids:
• DNA replication and repair
• Transcription
• Translation
• Ribosome synthesis
• RNA maduration and splicing
• Nuclear export processes
Eric J. On Helicases and other motor proeins. Cur Opin Stuct Biol. 2008
April; 18(2):243-257
1. Definition and Function
DNA vs RNA helicases
 Closely related in structure and sequence
 RNA – encoded by organisms from all kingdoms of life and by
many viruses.
 RNA helicases outnumber DNA helicases
Nomenclature for subfamilies:
Singleton MR, Dillngham MS. Structure and mechanism of
helicases and nucleic acid translocases. Annu. Rev. Biochem.
2007; 76:23-50.
2. Superfamilies (SF)
Classification based on the primary amino acid
sequences of the helicases.
Alfa Helicases



Beta Helicases



SF – 1
SF – 2
SF – 3
SF – 4
SF – 5
SF – 6
MONOMERIC
A & B helicases
A helicases
B helicases
B helicases
A & B helicases
HEXAMERIC
2. Superfamilies (SF)
SF1 & SF2
SF3 – SF6
General
Very prevalent
Monomeric
Form hexameric rings
Function
Several diverse DNA and
RNA manipulations
Replication fork
Domains
Two recA-like
RecA-like or
AAA+
ATP-binding site
At the interface of these
two domains
Consists of elements derived
from monomers in the
complex
2. Superfamilies (SF)
Fairman-Williams ME, Guenther U, Jankowsky E. SF1 and SF2
helicases: family matters. Curr Opin Struct Biol. 2010 June;
20(3):313-324
Patel SS, Picha KM. Structure and function of hexameric
helicases. Annu. Rev. Biochem. 2000; 69:651-97
3. Hexameric helicases
•Why a ring?
Increases the processivity of their respective DNA
polymerases
• High processivity is needed to catalyze genome replication and
recombination processes.
By encircling the DNA, helicases are topologically
linked to the DNA
• It allows helicases to stay on the DNA longer.
It decrease the probability of complete
dissociation from the DNA
3. Hexameric helicases
Types
DNA or RNA
Direction of
unwinding
Examples
Bacteriophage
Helicases
ssDNA
5’-3’
- T7 gp4 Proteins
-T4 gp41 Protein
- SPP1 G40P Protein
Plasmid-Encoded
Helicase
ssDNA
5’-3’
RSF1010 RepA Protein
Bacterial Helicases
ssDNA/ssRNA
5’-3’
- E.Coli DnaB Protein
-E.coli RuvB Protein
- E.coli rho Protein
Archaeal Helicase
ssDNA
3’-5’
Methanobacterium
thermoautotrophicum MCM
3’-5’
-SV40 and Polyoma Large
T Antigen Proteins
- Papillomavirus E1 Protein
3’-5’
-Human Bloom’s Syndrome
protein
- Mammalian MCM 4,6,7
Eukaryotic Viral
Helicases
Eukaryotic
Helicases
dsDNA
ssDNA
4. Domains
• RecA-like
Jiqing Y, Osborne AR. RecA-like motor ATPases – lessons from
structures. Biochemica et Biophysica Acta. 2004; 1-18
• AAA+
5. Evolution of DnaB helicase
DnaB originated from a duplication
of RecA-like ancestor after the
divergence of the bacteria from
Archaea and eukaryotes
The replication fork helicases in
Bacteria and
Archaea/Eukaryota have
evolved independently
Leipe DD, Aravind L. The bacterial replicative helicase DnaB evolved from
a RecA duplication. Cold Spring Harbor Laboratory Press ISSN. 2000; 10:516
MATERIALS AND METHODS
1.
2.
3.
4.
5.
Data bases
Sequence alignment
Structural aligment
Superimpositions
Display
1. Databases




PDB
Uniprot
SCOP
Pfam
2. Sequence aligment


T – coffee
Clustalw
T_coffee input.fa > output.fa
Clustalw input.fa > output.fa
Clustal format
3. Structural aligment

HMMER
HMM fetch
Target protein
HMM scan
PFAM family of
the target
HMM from
PFAM family
alignment
HMM align
Sequence –
based
alignment
4. Superimpositions

Rough STAMP
.out
INPUT
.domains
.pdb
(input)
Rough
STAMP
Transform
Chimera
.pdb
(output)
RMSD (Root mean Standard deviation)
Proteins
superimposed
5. Display

Chimera
PCR A
1. Structure
2. Motifs
3. Mechanism
1. Structure
4 structural domains:
2 α-β parallel domains (dominis 1a and 2a)
2 additional domains (1b and 2b)
Velankar SS, Soultanas P, Dillingham MS, Subramanya HS, Wigley DB. Crystal
Structures of Complexes of PcrA DNA Helicase with DNA Substrate Indicate an
Inchworm Mechanism. Cell 1999, 97 (75-84)
1. Structure
RecA-like core
Caruthers MJ, McKay D. Helicase structure and mechanism. Current opinion in Structural Biology 2002, 12: 123-133
1. Structure
Caruthers MJ, McKay D. Helicase structure and mechanism. Current opinion in Structural Biology 2002, 12: 123-133
2. Motifs
Walker A
Walker B
• Motif I (Walker A):
o
Amino group of lysine interacts with phosphates of MgATP/MgADP
o
Hydroxyl of serine or threonine coordinates Mg2+ ion
• Motif II (Walker B):
o D227 forms salt bridge with K568 of motif V
2. Motifs
• Walker A
Walker A (I) and Walker B (II)
Lys37-ATP
• Walker B
• Motif I (Walker A):
o
Amino group of lysine interacts with
phosphates of MgATP/MgADP
2. Motifs
• Motif II (Walker B):
o D227 forms salt bridge with K568 of motif V
Asp227
Lys568
2. Motifs
• Motif Ia :
o
Backbone carbonyl of F64 hydrogen bonds with ribose hydroxyl of ssDNA
• Motif IV:
o
R359 binds DNA and forms a salt bridge to E600
o
N361 interacts with ssDNA
• Motif TxGx:
o
T91 and H93 interact with terminal nucleotide on ssDNA.
2. Motifs
Ia, IV and TxGx
• Ia
• IV
• TxGx
2. Motifs
• Motif Ia :
o Backbone carbonyl of F64 hydrogen bonds with ribose hydroxyl of ssDNA
Hydrogen bond
Phe64
2. Motifs
• Motif IV:
o N361 interacts with ssDNA
Asn361
2. Motifs
• Motif IV:
o R359 binds DNA and forms a salt bridge to E600
Arg359
Glu600
2. Motifs
• Motif III :
o
D251 and D253 form salt bridges with K309 and R206 respectively
o
Q254 interacts with γ phosphate of ATP
o
Y257, W259 and R260 interact with oligonucleotide
• Motif V:
o
H565 interacts with ssDNA
o
K568 forms salt bridge with E224 and D227 of motif II
o
E571 interacts with ribose of ATP
2. Motifs
III and V
• III
• V
2. Motifs
• Motif III :
o
D251 and D253 form salt bridges with K309 and R306 respectively
Asp251
Arg306
Asp253
Lys309
2. Motifs
Caruthers MJ, McKay D. Helicase structure and mechanism. Current opinion in Structural Biology 2002, 12: 123-133
2. Motifs
• Motif V:
o
K568 forms salt bridge with E224 and D227 of motif II
Glu224
Lys568
Asp227
2. Motifs
Caruthers MJ, McKay D. Helicase structure and mechanism. Current opinion in Structural Biology 2002, 12: 123-133
2. Motifs
• Motif VI :
o
R610 interacts with γ phosphate of ATP
2. Motifs
VI
2. Motifs
• Motif VI:
o
R610 interacts with γ phosphate of ATP
3. Mechanism
Active Rolling
Inchworm
• Requirement for a dimeric
protein
• Consistent with any oligomeric
• Each subunit binds to ssDNA or
dsDNA but not at the same time
• Binding to both ssDNA and dsDNA
at the same time
• Large step sizes
• Smaller step sizes
state of the protein
Velanker SS, Soultnas P, Dillingham MS, Subramanya HS, Wigley DB. Crystal structures of complexes of PcrA DNA helicase with a
DNA substrate indicate an inchworm mechanism. Cell. 1999 Apr 2; 97 (1): 75-84.
3. Mechanism
Active Rolling
Inchworm
Patel SS, Donmex I. Mechanism of helicases. J Biol Chem. 2006 Jul 7; 281 (27).
DNA B
1.
2.
3.
4.
Introduction
Structure
Motifs
Mechanism
1. Introduction
SUPER FAMILY 4
DnaB family
DnaB
RepA
T4 and T7
bacteriophages
2. Structure
Ring – shaped hexameric helicasa
6 identical monomers
2 structural domains:
• 1 α-β domain = CTD
• 1 α domain = NTD
• conected by a linker
2. Structure
CTD
NTD
3. Motifs
Hall MC, Matson SW. Helicase motifs: the engine that powers DNA unwinding. Molecular Microbiology. 1999; 34(5): 867-877.
Bailey S, Eliason WK, Steitz TA. The crystal structure of the Thermus aquaticus DnaB helicase monomer. Nucleic Acids Research. 2007; 35(14): 4728-36.
3. Motifs
Walker A
Walker B
3. Motifs
Contacts with GDP
Walker A
GLY 215
LYS 216
THR 217
3. Motifs
H1a
H2 (Walker B)
3. Motifs
H1a
H2 (Walker B)
ASP 320
GLU 241
3. Motifs
H3
3. Motifs
H3
GLN 362
4. Mechanism
Brownian motor
Stepping
• One nucleic acid binding site
• Two nucleic acid binding sites
• Two conformational changes,
tight state and weak state
• Six conformacional changes, for
each subunit
• Power stroke motion + brownian
motion
• Power stroke motion
4. Mechanism
Brownian motor
Stepping
ALIGNMENTS
1. PcrA
a. Sequence alignment
b. Structural alignment
c. Superimposition
2. DnaB
a. Sequence alignment
b. Structural alignment
c. Superimposition
3. Helicases
a. Sequence alignment
b. Structural alignment
c. Superimposition
1. PcrA


Program: T – coffee
Templates:
Uniprot ID
Organism
C3QZ11
Bacteriodes sp.
O34580
Bacillus Subtilis
P9WNP4
Mycobacterium tuberculosis
P56255
Geobacillus stearothermophilus
Q3DRY9
Streptococcus agalactiae
Q8CRT9
Straphylococcus epidermidis
Q9S3Q0
Leuconostroc citreum
Q53727
Straphylococcus aureus
1. PcrA
Sequence alignment
Walker B
Walker A
1. PcrA
Structural alignment
Conserved
N - terminal
Non conserved
C - terminal
1. PcrA
Superimposition PcrA vs PcrA+ATP
-
-
PcrA (1PJR)
PcrA + ATP (3PJR)
DNA
ATP
RMSD: 2’33
Sc: 4’27
2. DnaB


Program:
T – coffee
Templates:
Uniprot ID
Organism
A1AIN1
Escherichia coli
O78411
Guillardia theta
P59966
Mycobacterium bovis
Q55418
Synechocystis sp.
O30477
Rhodothermus marinus
P0A1Q4
Salmonella typhimurium
P47340
Mycoplasma genitalium
P75539
Mycoplasma pneumoniae
Q8YZA1
Nostoc sp.
P45256
Haemophilus influenzae
P51333
Prophyra purpurea
P9WMR2
Mycobacterium tuberculosi
Q9X4C9
Geobacillus stearothermophilus
2. DnaB
Sequence alignment
Walker A
Walker B
2. DnaB
Structural alignment
Non conserved
N - terminal
Conserved
C - terminal
3. Helicases


Program:
Clustalw
Templates:
PDB ID
Organism
Type of helicase
1E0K
Enterobacteria phage
T7
Hexamer
2REB
Escherichia coli
Monomeric
1PJR
Geobacillus
stearothermophilus
Monomeric
4ESV
Geobacillus
stearothermophilus
Hexameric
1A1V
Hepatitis c virus
Monomeric
1FUU
Saccharomyces
cerevisiae
Monomeric
1PV4
Escherichia coli
Hexameric
1UAA
Escherichia coli
Monomeric
1D2M
Thermus thermophilus
Monomeric
3. Helicases
Sequence alignment
Walker A
Walker B
3. Helicases
Helicase
PDB
ID
Family
Superfam
Domains
Organism
Pfam
Pfam’s
code
RecA
2REB
RecA proteinlike (ATPase-domain)
P-loop containing
nucleoside triphosphate
hydrolases
a: 3-268
Escherichia coli
(strain K12)
RecA
PF00154
DnaB
4ESV
a: 183-441
Geobacillus
stearothermophil
us
DnaB
PF00772
HCV
1A1V
P-loop containing
nucleoside triphosphate
hydrolases
a: 190-325
Hepatitis C virus
genotype 1ª
(isolate H)
HCV
core
PF01542
Tandem
AAA-ATPase
domain
P-loop containing
nucleoside triphosphate
hydrolases
a: 11-225
Saccharomyces
cerevisiae (strain
ATCC
204508/S288c
) (Baker’s yeast)
Helicas
e Ctermina
l
domain
PF00271
RecA proteinlike (ATPasedomain)
P-loop containing
nucleoside triphosphate
hydrolases
Escherichia coli
(strain K12)
Rho Ntermina
l
domain
PF07498
IF4A
Rho
1FUU
1PV4
RNA helicase
b: 326-624
b: 226-394
a-f: 129-417
3. Helicases
Helicase
PDB
ID
Family
Superfam
Domains
Organism
Pfam
Pfam’s
code
Rep
1UAA
Tandem
AAA- ATPase
domain
P-loop containing
nucleoside triphosphate
hydrolases
a: 2-307
Escherichia coli
(strain K12)
UvrD
helicas
e
PF00580
Tandem
AAA- ATPase
domain
P-loop containing
nucleoside triphosphate
hydrolases
Thermus
thermophilus
(strain
HBB/ATCC
27634/DSM
579)
UvrB
PF12344
UvrB
1D2M
b: 308-640
a: 2-409
b: 410-583
T7
1E0K
RecA proteinlike (ATPase
domain)
P-loop containing
nucleoside triphosphate
hydrolases
chains a-f
Bacteriophage
T7
DnaB_
C
PF03796
PcrA
1PJR
Tandem
AAA- ATPase
domain
P-loop containing
nucleoside triphosphate
hydrolases
a: 1-318
Bacillus
stearothermophi
lus
UvrDhelicas
e
PF00580
b: 319-651
3. Helicases
Structural alignment: PDB ID
N – terminal conserved
C – terminal conserved
1PJR
1E0K
1FUU
4ESV
1UAA
2REB
1D2M
1PV4
3. Helicases
Structural alignment: N – terminal domain
Non-conserved
Conserved
Non-conserved
3. Helicases
Structural alignment: C – terminal domain
Non-conserved
Conserved
Non-conserved
3. Helicases
Superimposition PcrA - Rep
-
PcrA (1PJR)
Rep (1UAA)
RMSD: 1’39
Sc: 4’53
CONCLUSIONS



Helicases are essential proteins for every living
organism.
The ATP-binding motifs (Walker A and Walker B)
are highly conserved.
The important structures are mantained along
evolution in order to preserve the function of the
enzymes.
BIBLIOGRAPHY
• Enemark EJ, Tor LJ. On helicases and other motor proteins. Curr Opin Struct Biol. 2008 April; 18(2): 243-257.
• Singleton MR, Dillingham MS, Wigley DB. Structure and mechanism of helicases and nucleic acid translocases. Annu
Rev Biochem. 2007; 76: 23-50.
• Hall MC, Matson SW. Helicase motifs: the engine that powers DNA unwinding. Molecular Microbiology. 1999; 34(5):
867-877.
• Patel SS, Picha KM. Structure and function of hexameric helicases. Annu Rev Biochem. 2000; 69: 651-97.
• Fairman-Williams ME, Guenther UP, Jankowsky E. SF1 and SF2 helicases: family matters. Curr Opin Struct Biol. 2010
June; 20(3): 313-324.
• Leipe DD, Aravind L,Grishin NV, Koonin EV. The Bacterial replicative helicase DnaB evolved from a RecA duplication.
Genome Res. 2000 Jan; 10(1): 5-16.
• Neuwald AF, Aravind L, Spouge JL, Koonin EV. AAA+: A class of chaperone-like ATPases associated with the assembly,
operation, and disassembly of protein complexes. Genome Res. 1999 Jan; 9(1): 27-43.
• Ye J, Osborne AR, Groll M, Rapoport TA. RecA-like motor ATPases –lessons from structures. Biochim Biophys Acta.
2004 Nov 4; 1659(1): 1-18.
• Soultanas P, Wigley DB. Site-directed mutagenesis reveals roles for conserved amino acid residues in the hexameric
DNA helicase DnaB from Bacillus stearothermophilus. Nucleic Acids Research. 2002; 30: 4051-4060.
• Bailey S, Eliason WK, Steitz TA. The crystal structure of the Thermus aquaticus DnaB helicase monomer. Nucleic Acids
Research. 2007; 35(14): 4728-36.
• Soultanas P. Loading mechanisms of ring helicases at replication origins. Mol Microbiol. 2012 Apr; 84(1): 6-16.
• Bailey S, Eliason WK, Steitz TA. Structure of hexameric DnaB helicase and its complex with a domain of DnaG
primase. Science. 2007 Oct; 318(5849): 459-63.
• Itsathitphaisarn O, Wing RA, Eliason WK, Wang J. The non-planar structure of DnaB hexamer with its substrates
suggests a different mechanisms of translocation. Cell. 2012 Oct 12; 151(2): 267-277.
QUESTIONS
1. In which processes are helicases involved?
a. DNA replication
b. Ribosome synthesis
c. Nuclear export processes
d. DNA repair
e. All are correct
2. Helicases are classified in six superfamilies:
a. Hexameric helicases are SF3, SF4, SF5 and SF6
b. Monomeric helicases are SF3, SF4, SF5 and SF6
c. SF1 and SF2 are hexameric helicases
d. All superfamilies are hexameric helicases
e. All superfamilies are monomeric helicases
QUESTIONS
3. PcrA:
a. The organisms that have it are gram-negative bacteria
b. Belongs to SF1
c. A and B are correct
d. Hexameric helicase
e. All are correct
4. Motifs of PcrA:
a. Walker A is motif I and Walker B is motif II
b. Walker A is the only motif
c. Walker A interacts with DNA
d. Walker B interacts with DNA
e. All are correct
QUESTIONS
5. DnaB:
a. Hexameric helicase
b. Bacterial helicase
c. A and B are correct
d. It doesn’t have B – sheet folds
e. All are correct
6. Motifs of DnaB:
a. Are located at the C-terminal part of DnaB
b. DnaB has 5 conserved motifs
c. A and B are correct
d. The N-terminal part of DnaB is conserved
e. All are correct
QUESTIONS
7. About PcrA and DnaB helicases:
a. PcrA belongs to SF1
b. DnaB belongs to SF4
c. PcrA participates in the replication of some plasmids
d. DnaB is the main replicative helicase of eubacteria kingdom
e. All are correct
8. When aligning PcrA helicases from different organisms:
a. Walker A motif is conserved
b. Walker A motif is not conserved
c. The N-terminal domain is not structurally conserved
d. The C-terminal domain is structurally conserved
e. Any motif is conserved
QUESTIONS
9. When aligning DnaB helicases from different organisms:
a. Walker A and Walker B motifs are conserved
b. N-terminal domain is structurally conserved
c. C-terminal domain is structurally conserved
d. A and B are correct
e. A and C are correct
10. When aligning different types of helicases:
a. The N-terminal domain always contains the Walker A motif
b. The C-terminal domain always contains the Walker B motif
c. A and B are correct
d. DNA and ATP-binding motifs are conserved among
helicases
e. All are correct
Thank you!