Helicases: Methods and Protocols

402 Pages·2010·8.229 MB·English

by  Mohamed Abdelhaleem (auth.), Mohamed M. Abdelhaleem (eds.)

Springer Protocols

Methods in Molecular Biology 587
Helicases
Methods and Protocols
Edited by
Mohamed M. Abdelhaleem
M M B TM
ETHODS IN OLECULAR IOLOGY
SeriesEditor
JohnM.Walker
SchoolofLifeSciences
UniversityofHertfordshire
Hatfield,Hertfordshire,AL109AB,UK
Forothertitlespublishedinthisseries,goto
www.springer.com/series/7651
Helicases
Methods and Protocols
Edited by
Mohamed M. Abdelhaleem
University of Toronto, Toronto, ON, Canada
Editor
MohamedM.Abdelhaleem
HospitalforSickChildren
DepartmentofPaediatricLaboratory
Medicine
555UniversityAve.
TorontoONM5G1X8
Canada
[email protected]
ISSN1064-3745 e-ISSN1940-6029
ISBN978-1-60327-354-1 e-ISBN978-1-60327-355-8
DOI10.1007/978-1-60327-355-8
LibraryofCongressControlNumber:2009936373
#HumanaPress,apartofSpringerScienceþBusinessMedia,LLC2010
Allrightsreserved.Thisworkmaynotbetranslatedorcopiedinwholeorinpartwithoutthewrittenpermissionofthe
publisher (Humana Press,c/o Springer ScienceþBusinessMedia, LLC, 233 Spring Street, New York, NY 10013,
USA),exceptforbriefexcerptsinconnectionwithreviewsorscholarlyanalysis.Useinconnectionwithanyformof
informationstorageandretrieval,electronicadaptation,computersoftware,orbysimilarordissimilarmethodology
nowknownorhereafterdevelopedisforbidden.
Theuseinthispublicationoftradenames,trademarks,servicemarks,andsimilarterms,eveniftheyarenotidentifiedas
such,isnottobetakenasanexpressionofopinionastowhetherornottheyaresubjecttoproprietaryrights.
Printedonacid-freepaper
springer.com
Preface
The objective of this book is to provide the scientific community with the current
methods used to study helicases, the enzymes that utilize the energy derived from
nucleoside triphosphate (NTP) hydrolysis to unwind the double-stranded helical
structure of nucleic acids. The book starts with an overview chapter that provides a
briefintroductionofhelicases,withfewexamplesoftheirroleinnucleicacidmetabo-
lism.Thischapterisintendedforreadersnewtothefield.Thechaptersthatfolloware
writtenbyleadinginternationalscientistswhocontributedsignificantlytoourcurrent
understandingofhelicases.Inthesechapters,thereaderfindsmethodsfortheproduc-
tion and purification of helicases from different species as well as detailed studies of
helicase activities, including NTP binding and hydrolysis, nucleic acid binding and
unwinding,andtranslocationalongnucleicacidsubstrates.
Helicase activities are generally measured by methods that rely on radiometric,
enzymatic, and fluorescence-based techniques. As described in Chapter 2, fluores-
cenceprobeshavehighsensitivityandrapidresponsethatpermitrealtimeanalysis.The
abilitytousefluorescenceprobestoinvestigatehelicaseactivityatthesinglemolecule
level has provided significant insights into the mechanism of helicase function. A
generalguidetosuchassaysisgiveninChapter3.Anotheradvanceinhelicasestudies
istheuseofpre-steadystatekinetictechniques,asdescribedforthetranslocaseactivity
along single-stranded nucleic acids (Chapter 4) and for the DNA unwinding and
polymerization activities of bacteriophage T7 molecule (Chapter 5). In addition to
nucleicacidunwinding,helicasesareinvolvedinthedisruptionofprotein–nucleicacid
interactions.Chapter6hasfourexamplesofproteindisplacementbyhelicasesinvolved
in various steps of nucleic acid metabolism. As helicases occur in vivo as part of
molecular complexes that include nucleic acid and protein, characterization of
their protein and nucleic acid interactions provides insights into their in vivo roles.
Chapter7describesaprotocoltoinvestigateprotein–proteininteractionsinvivousing
tandem affinity purification. Chapter8 describesthe use of chromatin immunopreci-
pitation(ChIP)todeterminenucleicacidtargetsofDNAhelicases.
The following three chapters describe methods to study DNA helicases involved in
replication (Chapter 9), transcription termination (Chapter 10), and recombination
(Chapter 11). The RecQ family of DNA helicases is involved in DNA repair and
recombination and has been the subject of intense research because of their roles in
maintaining genome stability. Three chapters are devoted to the study of RecQ heli-
cases from different species. A protocol for the production and characterization of
mutants of the helicase core of human Bloom syndrome gene (BLM) is described in
Chapter 12. This protocol can be adapted to study other helicases. Chapter 13
describestheroleofDrosophilaBlmhelicaseindouble-strandedgaprepair.Protocols
fortheexpressionandcharacterizationofRecQhelicasesfromtheplantmodelArabi-
dopsisthalianaaredescribedinChapter14.
v
vi Preface
The following chapters describe methods to study hepatitis C virus (HCV) non-
structuralprotein3(NS3),apotentialtherapeutictargetfortheliverdiseasecausedby
HCV. Chapter 15 describes a fluorescence-based high-throughput assay to test inhi-
bitors of NS3 helicase activity. In addition to the C-terminal helicase domain, NS3
protein has an N terminal serine protease. A method to simultaneously monitor the
helicaseandproteaseactivitiesofHCVNS3isdescribedinChapter16.Thismethod
could be used to identify dual NS3 inhibitors. In Chapter 17, computational techni-
questostudyNS3aredescribed.
RNA helicase members of the DExD/H-box family of proteins are involved in all
aspectsofcellularRNAmetabolism.Chapter18providesaprotocolforthequantita-
tive evaluation of the unwinding activity of the largest family of RNA helicase, the
DEAD-boxproteins.Examplesofthemethodsusedtostudytheversatilerolesplayed
by RNA helicases include the role of Ddx5 in transcription (Chapter 19), the role of
DDX3inHIVinfection(Chapter20),andtheactivitiesofDHX9(RNAhelicaseAand
nuclear DNA helicase II) (Chapter 21) and its drosophila homolog maleless protein
(Chapter 22). Chapter 23 describes cloning, expression and activities of the human
RNAhelicase,Upf1,whichisinvolvedinnon-sensemediateddecayofmRNA.
Helicases are involved in regulating the mitochondrial genome. Chapter 24 pro-
videsprotocolstostudytheactivitiesofthemitochondrialdegradosome,includingthe
activityof itshelicasecomponent,Suv3p. Chapter25describesthehelicaseandanti-
telomereactivitiesofPif1p,afunctionallyversatilehelicaseinvolvedinregulatingboth
themitochondrialandnucleargenomes.
Thelasttwochaptersillustratetwoapplicationsofhelicaseresearchinagricultureand
medicine.Chapter26describesamethodtoconfersalinitystresstolerancetoplantsby
overexpression of a DNA helicase. Chapter 27 describes the potential targeting of
helicasestoinhibitthegrowthofmalariaparasites.
This book has only been made possible by the contributions from the authors. I
would like to thank all of them for their cooperation and timely submissions. I am
grateful to the series editor, John Walker, for his helpful comments and editorial
expertise.Finally,Iwouldliketoacknowledgethesupportofmyfamily.
MohamedM.Abdelhaleem
Contents
Preface............................................................ v
Contributors........................................................ ix
1. Helicases:AnOverview.............................................. 1
MohamedAbdelhaleem
2. FluorescentBiosensorstoInvestigateHelicaseActivity ..................... 13
MartinR.Webb
3. Single-MoleculeFRETAnalysisofHelicaseFunctions...................... 29
EliRothenbergandTaekjipHa
4. KineticsofMotorProteinTranslocationonSingle-StrandedDNA ............ 45
ChristopherJ.Fischer,LakeWooten,EricJ.Tomko,andTimothyM.Lohman
5. ExperimentalandComputationalAnalysisofDNAUnwindingandPolymerization
Kinetics.......................................................... 57
ManjulaPandey,MikhailK.Levin,andSmitaS.Patel
6. ProteinDisplacementbyHelicases..................................... 85
LaxmiYeruvaandKevinD.Raney
7. InVivoInvestigationofProtein–ProteinInteractionsforHelicasesUsing
TandemAffinityPurification.......................................... 99
MatthewJessulat,TerryBuist,MdAlamgir,MohsenHooshyar,JianhuaXu,
HiroyukiAoki,M.CleliaGanoza,GarethButland,andAshkanGolshani
8. MappingGenomicTargetsofDNAHelicasesbyChromatin
ImmunoprecipitationinSaccharomycescerevisiae .......................... 113
JenniferCobbandHaicovanAttikum
9. MethodstoStudyHowReplicationForkHelicasesUnwindDNA ............ 127
DanielL.KaplanandIrinaBruck
10. SimpleEnzymaticAssaysfortheInVitroMotorActivityofTranscription
TerminationFactorRhofromEscherichiacoli............................. 137
MarcBoudvillain,Ce´lineWalmacq,AnnieSchwartz,
andFre´de´riqueJacquinot
11. Single-MoleculeStudiesofRecBCD.................................... 155
ThomasT.PerkinsandHung-WenLi
12. MutationalAnalysisofBloomHelicase.................................. 173
XuGuangXi
13. InVivoAnalysisofDrosophilaBLMHelicaseFunctionDuringDNA
Double-StrandGapRepair........................................... 185
MitchMcVey
14. PurificationandCharacterizationofRecQHelicasesofPlants................ 195
DanielaKobbe,ManfredFocke,andHolgerPuchta
vii
viii Contents
15. FluorometricAssayofHepatitisCVirusNS3HelicaseActivity ............... 211
MariuszKrawczyk,AnnaStankiewicz-Drogon´,Anne-LiseHaenni,
andAnnaBoguszewska-Chachulska
16. AMethodtoSimultaneouslyMonitorHepatitisCVirusNS3Helicase
andProteaseActivities .............................................. 223
DavidN.Frick,OlyaGinzburg,andAngelaM.I.Lam
17. ComputerModelingofHelicasesUsingElasticNetworkModel .............. 235
WenjunZheng
18. DuplexUnwindingwithDEAD-BoxProteins ............................ 245
EckhardJankowskyandAndreaPutnam
19. AnalysisoftheRNAHelicasep68(Ddx5)asaTranscriptionalRegulator ....... 265
SamanthaM.NicolandFrancesV.Fuller-Pace
20. AMethodtoStudytheRoleofDDX3RNAHelicaseinHIV-1 .............. 281
Chia-YenChen,VenkatR.K.Yedavalli,andKuan-TehJeang
21. MolecularCharacterizationofNuclearDNAHelicaseII(RNAHelicaseA) ..... 291
SuishengZhangandFrankGrosse
22. RegulationofInter-andIntramolecularInteractionofRNA,DNA,andProteins
byMLE.......................................................... 303
HyangyeeOh,AndrewM.Parrott,YongkyuPark,andChee-GunLee
23. BiochemicalCharacterizationofHumanUpf1Helicase..................... 327
ZhihongCheng,GakuMorisawa,andHaiweiSong
24. AssaysoftheHelicase,ATPase,andExoribonucleaseActivitiesoftheYeast
MitochondrialDegradosome ......................................... 339
MichalMalecki,PiotrP.Stepien,andPawelGolik
25. CharacterizationoftheHelicaseActivityandAnti-telomerasePropertiesofYeast
Pif1pInVitro..................................................... 359
Jean-BaptisteBoule´ andVirginiaA.Zakian
26. AMethodtoConferSalinityStressTolerancetoPlantsbyHelicase
Overexpression.................................................... 377
NarendraTuteja
27. AMethodtoInhibittheGrowthofPlasmodiumfalciparumbyDouble-Stranded
RNA-MediatedGeneSilencingofHelicases.............................. 389
RenuTuteja
SubjectIndex .......................................................... 401
Contributors
MOHAMEDABDELHALEEM (cid:129) DepartmentofPaediatricLaboratoryMedicine,TheHospi-
talforSickChildren,UniversityofToronto,Toronto,ON,Canada
MDALAMGIR (cid:129) DepartmentofBiologyandOttawaInstituteofSystemsBiology,Carleton
University,Ottawa,ON,Canada
HIROYUKI AOKI (cid:129) Banting and Best Department of Medical Research, University of
Toronto,Toronto,ON,Canada
ANNABOGUSZEWSKA-CHACHULSKA (cid:129) Genomed,Warsaw,Poland
MARCBOUDVILLAIN (cid:129) CentredeBiophysiqueMoleculaire(UPR4301),CNRS,Orleans
cedex2,France
JEAN-BAPTISTE BOULE (cid:129) Department of Molecular Biology, Princeton University, Prin-
ceton,NJ,USA
IRINABRUCK (cid:129) DepartmentofBiologicalSciences,VanderbiltUniversityNashville,TN,
USA
TERRYBUIST (cid:129) DepartmentofBiologyandOttawaInstituteofSystemsBiology,Carleton
University,Ottawa,ON,Canada
GARETH BUTLAND (cid:129) Life Science Division, Lawrence Berkeley National Laboratory,
Berkeley,CA,USA
CHIA-YEN CHEN (cid:129) Molecular Virology Section, Laboratory of Molecular, Microbiology,
theNIAID,NIH,Bethesda,MD,USA
ZHIHONGCHENG (cid:129) CancerandDevelopmentalCellBiologyDivision,InstituteofMole-
cular and Cell Biology, A*STAR (Agency for Science, Technology and Research),
Singapore,Singapore
JENNIFERCOBB (cid:129) DepartmentofBiochemistryandMolecularBiology,SouthernAlberta
CancerResearchInstitute,UniversityofCalgary,Calgary,AB,Canada
CHRISTOPHERJ.FISCHER (cid:129) DepartmentofPhysicsandAstronomy,UniversityofKansas,
Lawrence,KS,USA
MANFREDFOCKE (cid:129) BotanikII,Universita¨tKarlsruhe(TH),Karlsruhe,Germany
FRANCES V. FULLER-PACE (cid:129) Centre for Oncology & Molecular Medicine, University of
DundeeNinewellsHospital&MedicalSchool,Dundee,UK
DAVIDN.FRICK (cid:129) DepartmentofBiochemistry&MolecularBiologyNewYorkMedical
CollegeValhalla,NY,USA
M.CLELIAGANOZA (cid:129) BantingandBestDepartmentofMedicalResearch,Universityof
Toronto,Toronto,ON,Canada
OLYAGINZBURG (cid:129) DepartmentofBiochemistry&MolecularBiology,NewYorkMedical
College,Valhalla,NY,USA
PAWELGOLIK (cid:129) FacultyofBiology,InstituteofGeneticsandBiotechnology,Universityof
Warsaw,Warsaw,Poland
ASHKAN GOLSHANI (cid:129) Department of Biology and Ottawa Institute of Systems Biology,
CarletonUniversity,Ottawa,ON,Canada
ix
x Contributors
FRANK GROSSE (cid:129) Leibniz Institute for Age Research, Fritz Lipmann Institute (FLI),
Jena,Germany
TAEKJIPHA (cid:129) DepartmentofPhysics,CenterforBiophysicsandComputationalBiology,
UniversityofIllinoisatUrbana-Champaign,Urbana,IL,USA
ANNE-LISEHAENNI (cid:129) InstitutJacquesMonod,Paris,France
MOHSEN HOOSHYAR (cid:129) Department of Biology and Ottawa Institute of Systems Biology,
CarletonUniversity,Ottawa,ON,Canada
ECKHARD JANKOWSKY (cid:129) Department of Biochemistry & Center for RNA Molecular
Biology,SchoolofMedicine,CaseWesternReserveUniversity,Cleveland,OH,USA
FREDERIQUE JACQUINOT (cid:129) Centre de Biophysique Moleculaire (UPR4301), CNRS,
Orleanscedex2,France
KUAN-TEH JEANG (cid:129) Molecular Virology Section, Laboratory of Molecular, Microbiology,
theNIAID,NIH,Bethesda,MD,USA
MATTHEW JESSULAT (cid:129) Department of Biology and Ottawa Institute of Systems Biology,
CarletonUniversity,Ottawa,ON,Canada
DANIELL.KAPLAN (cid:129) VanderbiltUniversityDepartmentofBiologicalSciencesNashville,
TN,USA
DANIELAKOBBE (cid:129) BotanikII,Universita¨tKarlsruhe(TH),Karlsruhe,Germany
MARIUSZ KRAWCZYK (cid:129) Institute of Biochemistry and Biophysics, Polish Academy of
Sciences,Warsaw,Poland
ANGELA M. I. LAM (cid:129) Department of Biochemistry & Molecular Biology, New York
MedicalCollege,Valhalla,NY,USA
CHEE-GUNLEE (cid:129) UMDNJ-NewJerseyMedicalSchool,DepartmentofBiochemistryand
MolecularBiology,Newark,NJ,USA
HUNG-WEN LI (cid:129) Department of Chemistry, National Taiwan University, Taipei
10617,Taiwan
TIMOTHY M. LOHMAN (cid:129) Department of Biochemistry and Molecular Biophysics,
WashingtonUniversitySchoolofMedicine,St.Louis,MO,USA
MICHALMALECKI (cid:129) InstituteofGeneticsandBiotechnology,FacultyofBiology,Univer-
sityofWarsaw,Warsaw,Poland
MIKHAIL K. LEVIN (cid:129) Department of Biostatistics & Bioinformatics, Duke University
MedicalCenter,Durham,NC,USA
MITCHMCVEY (cid:129) DepartmentofBiology,TuftsUniversity,Medford,MA
GAKUMORISAWA (cid:129) CancerandDevelopmentalCellBiologyDivision,InstituteofMole-
cular and Cell Biology, A*STAR (Agency for Science, Technology and Research),
Singapore,Singapore
SAMANTHAM.NICOL (cid:129) CentreforOncology&MolecularMedicine,UniversityofDun-
deeNinewellsHospital&MedicalSchool.Dundee,UK
HYANGYEE OH (cid:129) HHMI, Waksman Institute, Rutgers University, Piscataway, NJ,
USA
MANJULA PANDEY (cid:129) Department of Biochemistry, Universityof Medicineand Dentistry
ofNewJersey-RobertWoodJohnsonMedicalSchool,Piscataway,NJ,USA
SMITAS.PATEL (cid:129) DepartmentofBiochemistry,UniversityofMedicineandDentistryof
NewJersey-RobertWoodJohnsonMedicalSchool,Piscataway,NJ,USA
YONGKYU PARK (cid:129) UMDNJ-New JerseyMedical School, Department of Cell Biologyand
MolecularMedicine,NewarkNewJersey,USA