HELICASES

Batlle Masó, Laura

Rosich Sangrà, Elena

Sumarroca Bordas, Marina

Torrecilas Testa, Tatiana

INDEX

A. IntroductionB. Materials and methodsC. Monomeric helicase: PcrAD. Hexameric helicase: DnaBE. Alignments and SuperimpositionsF. Conclusion

INTRODUCTION

1. Definition and function2. Superfamilies3. Hexameric helicases4. RecA like and AAA+ domains5. 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)

SF – 1 SF – 2 SF – 3 SF – 4 SF – 5 SF – 6

MONOMERICA & B helicases

HEXAMERIC

A helicasesB helicasesB helicases

Alfa Helicases

Beta Helicases

Classification based on the primary amino acid sequences of the helicases.

A & B helicases

2. Superfamilies (SF)

SF1 & SF2 SF3 – SF6

General Very prevalentMonomeric Form hexameric rings

FunctionSeveral diverse DNA

and RNA manipulations

Replication fork

Domains Two recA-like RecA-like orAAA+

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

•Why a ring?

• High processivity is needed to catalyze genome replication and recombination processes.

Increases the processivity of their respective DNA polymerases

• It allows helicases to stay on the DNA longer.

By encircling the DNA, helicases are topologically linked to the DNA

It decrease the probability of complete dissociation from the

DNA

3. Hexameric helicases

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

Eukaryotic Viral Helicases dsDNA 3’-5’

-SV40 and Polyoma Large T Antigen Proteins- Papillomavirus E1 Protein

Eukaryotic Helicases ssDNA 3’-5’

-Human Bloom’s Syndrome protein- Mammalian MCM 4,6,7

4. Domains

• AAA+• RecA-like

Jiqing Y, Osborne AR. RecA-like motor ATPases – lessons from structures. Biochemica et Biophysica Acta. 2004; 1-18

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; 10:5-16

MATERIALS AND METHODS

1. Data bases2. Sequence alignment3. Structural aligment4. Superimpositions5. Display

1. Databases

PDB Uniprot SCOP Pfam

2. Sequence aligment

T – coffee Clustalw

Clustal format

T_coffee input.fa > output.fa

Clustalw input.fa > output.fa

3. Structural aligment

HMMER

Target protein

PFAM family of the target

HMM from PFAM family

alignment

Sequence – based

alignment

HMM fetch

HMM alignHMM scan

4. Superimpositions

Rough STAMP

RMSD (Root mean Standard deviation)

.domains Rough STAMP

Transform Chimera

Proteins superimpos

ed

INPUT.out

.pdb(output

)

.pdb(input)

5. Display

Chimera

PCR A

1. Structure2. Motifs3. 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)

RecA-like core

1. Structure

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

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 (I) and Walker B (II)• Walker A

• Walker B

• Motif I (Walker A): o Amino group of lysine

interacts with phosphates of MgATP/MgADP

Lys37-ATP

2. Motifs

• Motif II (Walker B):o D227 forms salt bridge with K568 of motif V

Lys568

Asp227

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

Asp253Lys309

Arg306

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

2. Motifs

Lys568

Glu224

Asp227

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

2. Motifs

• Motif VI:o R610 interacts with γ phosphate of ATP

3. Mechanism

Active Rolling Inchworm

• Requirement for a dimeric protein

• Each subunit binds to ssDNA or dsDNA but not at the same time

• Large step sizes

• Consistent with any oligomeric state of the protein

• Binding to both ssDNA and dsDNA at the same time

• Smaller step sizes

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. Introduction2. Structure3. Motifs4. Mechanism

HELICASES

SUPER FAMILY 4

DnaB family

RepA

T4 and T7 bacteriophag

es

DnaB

1. Introduction

Ring – shaped hexameric helicasa6 identical monomers

2 structural domains:• 1 α-β domain = CTD• 1 α domain = NTD• conected by a linker

2. Structure

NTDCTD

2. Structure

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

3. Motifs

GLY 215

LYS 216

THR 217

Walker A

H1a

H2 (Walker B)

3. Motifs

3. Motifs

ASP 320

GLU 241

H1a

H2 (Walker B)

3. Motifs

H3

3. Motifs

H3

GLN 362

4. Mechanism

Brownian motor Stepping

• One nucleic acid binding site

• Two conformational changes, tight state and weak state

• Power stroke motion + brownian motion

• Two nucleic acid binding sites

• Six conformacional changes, for each subunit

• Power stroke motion

4. Mechanism

Brownian motor Stepping

ALIGNMENTS

1.PcrAa. Sequence alignmentb. Structural alignmentc. Superimposition

2. DnaBd. Sequence alignmente. Structural alignmentf. Superimposition

3. Helicasesg. Sequence alignmenth. Structural alignmenti. Superimposition

1. PcrA

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

Program: T – coffee Templates:

1. PcrA

Sequence alignment Walker A

Walker B

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

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

Program: T – coffee

Templates:

2. DnaB

Sequence alignment Walker A

Walker B

Structural alignment

Non conserved N - terminal

Conserved C -

terminal

2. DnaB

3. Helicases

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

Program: Clustalw

Templates:

Sequence alignment Walker A

Walker B

3. Helicases

Helicase

PDB ID

Family Superfam Domains

Organism Pfam

Pfam’s

code

RecA 2REB RecA protein-

like (ATP-ase-

domain)

P-loop containing nucleoside

triphosphate hydrolases

a: 3-268 Escherichia coli (strain

K12)

RecA PF00154

DnaB 4ESV a: 183-441 Geobacillus stearotherm

ophilus

DnaB PF00772

HCV 1A1V RNA helicase

P-loop containing nucleoside

triphosphate hydrolases

a: 190-325

b: 326-624

Hepatitis C virus

genotype 1ª (isolate H)

HCV core

PF01542

IF4A 1FUU Tandem AAA-

ATPase domain

P-loop containing nucleoside

triphosphate hydrolases

a: 11-225

b: 226-394

Saccharomyces

cerevisiae (strain ATCC 204508/S288c) (Baker’s

yeast)

Helicase C-

terminal

domain

PF00271

Rho 1PV4 RecA protein-

like (ATPase-domain)

P-loop containing nucleoside

triphosphate hydrolases

a-f: 129-417

Escherichia coli (strain

K12)

Rho N-

terminal

domain

PF07498

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

b: 308-640

Escherichia coli (strain

K12)

UvrD helica

se

PF00580

UvrB 1D2M Tandem AAA-

ATPase domain

P-loop containing nucleoside

triphosphate hydrolases

a: 2-409

b: 410-583

Thermus thermophilu

s (strain HBB/ATCC

27634/DSM 579)

UvrB PF12344

T7 1E0K RecA protein-

like (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

b: 319-651

Bacillus stearotherm

ophilus

UvrD-helica

se

PF00580

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

Conserved

Non-conserved

Non-conserved

3. Helicases

Structural alignment: C – terminal domain

Conserved

Non-conserved

Non-conserved

3. Helicases

Superimposition PcrA - Rep

- PcrA (1PJR)- Rep (1UAA)

RMSD: 1’39Sc: 4’53

3. Helicases

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 replicationb. Ribosome synthesisc. Nuclear export processesd. DNA repaire. All are correct

2. Helicases are classified in six superfamilies:a. Hexameric helicases are SF3, SF4, SF5

and SF6b. Monomeric helicases are SF3, SF4, SF5 and

SF6c. SF1 and SF2 are hexameric helicasesd. All superfamilies are hexameric helicasese. All superfamilies are monomeric helicases

QUESTIONS

3. PcrA:a. The organisms that have it are gram-negative

bacteriab. Belongs to SF1c. A and B are correctd. Hexameric helicasee. All are correct

4. Motifs of PcrA:a. Walker A is motif I and Walker B is motif IIb. Walker A is the only motifc. Walker A interacts with DNAd. Walker B interacts with DNAe. All are correct

QUESTIONS

5. DnaB:a. Hexameric helicaseb. Bacterial helicasec. A and B are correctd. It doesn’t have B – sheet folds e. All are correct

6. Motifs of DnaB:a. Are located at the C-terminal part of DnaBb. DnaB has 5 conserved motifsc. A and B are correctd. The N-terminal part of DnaB is conservede. All are correct

QUESTIONS

7. About PcrA and DnaB helicases:a. PcrA belongs to SF1b. DnaB belongs to SF4c. PcrA participates in the replication of some

plasmidsd. DnaB is the main replicative helicase of

eubacteria kingdome. All are correct

8. When aligning PcrA helicases from different organisms:

a. Walker A motif is conservedb. Walker A motif is not conservedc. The N-terminal domain is not structurally

conservedd. The C-terminal domain is structurally

conservede. Any motif is conserved

QUESTIONS

9. When aligning DnaB helicases from different organisms:

a. Walker A and Walker B motifs are conservedb. N-terminal domain is structurally conservedc. C-terminal domain is structurally conservedd. A and B are correcte. A and C are correct

10. When aligning different types of helicases:a. The N-terminal domain always contains the

Walker A motifb. The C-terminal domain always contains the

Walker B motifc. A and B are correctd. DNA and ATP-binding motifs are

conserved among helicasese. All are correct

Thank you!