helicases batlle masó, laura rosich sangrà, elena sumarroca bordas, marina torrecilas testa,...
TRANSCRIPT
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HELICASES
Batlle Masó, Laura
Rosich Sangrà, Elena
Sumarroca Bordas, Marina
Torrecilas Testa, Tatiana
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INDEX
A. IntroductionB. Materials and methodsC. Monomeric helicase: PcrAD. Hexameric helicase: DnaBE. Alignments and SuperimpositionsF. Conclusion
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INTRODUCTION
1. Definition and function2. Superfamilies3. Hexameric helicases4. RecA like and AAA+ domains5. Evolution
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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
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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.
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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
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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
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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
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•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
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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
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4. Domains
• AAA+• RecA-like
Jiqing Y, Osborne AR. RecA-like motor ATPases – lessons from structures. Biochemica et Biophysica Acta. 2004; 1-18
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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
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MATERIALS AND METHODS
1. Data bases2. Sequence alignment3. Structural aligment4. Superimpositions5. Display
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1. Databases
PDB Uniprot SCOP Pfam
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2. Sequence aligment
T – coffee Clustalw
Clustal format
T_coffee input.fa > output.fa
Clustalw input.fa > output.fa
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3. Structural aligment
HMMER
Target protein
PFAM family of the target
HMM from PFAM family
alignment
Sequence – based
alignment
HMM fetch
HMM alignHMM scan
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4. Superimpositions
Rough STAMP
RMSD (Root mean Standard deviation)
.domains Rough STAMP
Transform Chimera
Proteins superimpos
ed
INPUT.out
.pdb(output
)
.pdb(input)
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5. Display
Chimera
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PCR A
1. Structure2. Motifs3. Mechanism
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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)
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RecA-like core
1. Structure
Caruthers MJ, McKay D. Helicase structure and mechanism. Current opinion in Structural Biology 2002, 12: 123-133
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1. Structure
Caruthers MJ, McKay D. Helicase structure and mechanism. Current opinion in Structural Biology 2002, 12: 123-133
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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
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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
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• Motif II (Walker B):o D227 forms salt bridge with K568 of motif V
Lys568
Asp227
2. Motifs
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• 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
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Ia, IV and TxGx
• Ia
• IV
• TxGx
2. Motifs
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• Motif Ia : o Backbone carbonyl of F64 hydrogen bonds with ribose
hydroxyl of ssDNA
Hydrogen bond
Phe64
2. Motifs
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• Motif IV:o N361 interacts with ssDNA
Asn361
2. Motifs
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• Motif IV:o R359 binds DNA and forms a salt bridge
to E600
Arg359
Glu600
2. Motifs
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• 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
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III and V
• III
• V
2. Motifs
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• Motif III : o D251 and D253 form salt bridges with K309 and R306
respectively
Asp251
Asp253Lys309
Arg306
2. Motifs
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Caruthers MJ, McKay D. Helicase structure and mechanism. Current opinion in Structural Biology 2002, 12: 123-133
2. Motifs
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• Motif V:o K568 forms salt bridge with E224 and D227 of motif
II
2. Motifs
Lys568
Glu224
Asp227
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Caruthers MJ, McKay D. Helicase structure and mechanism. Current opinion in Structural Biology 2002, 12: 123-133
2. Motifs
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• Motif VI : o R610 interacts with γ phosphate of ATP
2. Motifs
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VI
2. Motifs
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2. Motifs
• Motif VI:o R610 interacts with γ phosphate of ATP
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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.
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3. Mechanism
Active Rolling Inchworm
Patel SS, Donmex I. Mechanism of helicases. J Biol Chem. 2006 Jul 7; 281 (27).
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DNA B
1. Introduction2. Structure3. Motifs4. Mechanism
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HELICASES
SUPER FAMILY 4
DnaB family
RepA
T4 and T7 bacteriophag
es
DnaB
1. Introduction
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Ring – shaped hexameric helicasa6 identical monomers
2 structural domains:• 1 α-β domain = CTD• 1 α domain = NTD• conected by a linker
2. Structure
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NTDCTD
2. Structure
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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
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Walker A
Walker B
3. Motifs
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Contacts with GDP
3. Motifs
GLY 215
LYS 216
THR 217
Walker A
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H1a
H2 (Walker B)
3. Motifs
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3. Motifs
ASP 320
GLU 241
H1a
H2 (Walker B)
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3. Motifs
H3
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3. Motifs
H3
GLN 362
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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
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4. Mechanism
Brownian motor Stepping
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ALIGNMENTS
1.PcrAa. Sequence alignmentb. Structural alignmentc. Superimposition
2. DnaBd. Sequence alignmente. Structural alignmentf. Superimposition
3. Helicasesg. Sequence alignmenth. Structural alignmenti. Superimposition
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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:
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1. PcrA
Sequence alignment Walker A
Walker B
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1. PcrA
Structural alignment
Conserved
N - terminal
Non conserved
C - terminal
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1. PcrA
Superimposition PcrA vs PcrA+ATP
- PcrA (1PJR)- PcrA + ATP (3PJR)- DNA- ATP
RMSD: 2’33
Sc: 4’27
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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:
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2. DnaB
Sequence alignment Walker A
Walker B
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Structural alignment
Non conserved N - terminal
Conserved C -
terminal
2. DnaB
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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:
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Sequence alignment Walker A
Walker B
3. Helicases
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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
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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
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Structural alignment: PDB ID
N – terminal conserved
C – terminal conserved
1PJR 1E0K
1FUU 4ESV
1UAA 2REB
1D2M
1PV4
3. Helicases
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Structural alignment: N – terminal domain
Conserved
Non-conserved
Non-conserved
3. Helicases
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Structural alignment: C – terminal domain
Conserved
Non-conserved
Non-conserved
3. Helicases
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Superimposition PcrA - Rep
- PcrA (1PJR)- Rep (1UAA)
RMSD: 1’39Sc: 4’53
3. Helicases
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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.
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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.
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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
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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
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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
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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
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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
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Thank you!