Dynamics and Mechanism of DNA-Bending Proteins in Binding Site Recognition PDF
Preview Dynamics and Mechanism of DNA-Bending Proteins in Binding Site Recognition
Springer Theses Recognizing Outstanding Ph.D. Research Yogambigai Velmurugu Dynamics and Mechanism of DNA-Bending Proteins in Binding Site Recognition Springer Theses Recognizing Outstanding Ph.D. Research Aims and Scope The series “Springer Theses” brings together a selection of the very best Ph.D. theses from around the world and across the physical sciences. Nominated and endorsed by two recognized specialists, each published volume has been selected foritsscientificexcellenceandthehighimpactofitscontentsforthepertinentfield of research. For greater accessibility to non-specialists, the published versions includeanextendedintroduction,aswellasaforewordbythestudent’ssupervisor explainingthespecialrelevanceoftheworkforthefield.Asawhole,theserieswill provide a valuable resource both for newcomers to the research fields described, and for other scientists seeking detailed background information on special ques- tions.Finally,itprovidesanaccrediteddocumentationofthevaluablecontributions madebytoday’syoungergenerationofscientists. Theses are accepted into the series by invited nomination only and must fulfill all of the following criteria (cid:129) TheymustbewritteningoodEnglish. (cid:129) ThetopicshouldfallwithintheconfinesofChemistry,Physics,EarthSciences, EngineeringandrelatedinterdisciplinaryfieldssuchasMaterials,Nanoscience, ChemicalEngineering,ComplexSystemsandBiophysics. (cid:129) Theworkreportedinthethesismustrepresentasignificantscientificadvance. (cid:129) Ifthethesisincludespreviouslypublishedmaterial,permissiontoreproducethis mustbegainedfromtherespectivecopyrightholder. (cid:129) They must have been examined and passed during the 12 months prior to nomination. (cid:129) Each thesis should include a foreword by the supervisor outlining the signifi- canceofitscontent. (cid:129) The theses should have a clearly defined structure including an introduction accessibletoscientistsnotexpertinthatparticularfield. More information about this series at http://www.springer.com/series/8790 Yogambigai Velmurugu Dynamics and Mechanism of DNA-Bending Proteins in Binding Site Recognition Doctoral Thesis accepted by University of Illinois at Chicago, Chicago, Illinois, USA YogambigaiVelmurugu UniversityofIllinoisatChicago Chicago,IL,USA ISSN2190-5053 ISSN2190-5061 (electronic) SpringerTheses ISBN978-3-319-45128-2 ISBN978-3-319-45129-9 (eBook) DOI10.1007/978-3-319-45129-9 LibraryofCongressControlNumber:2016952635 ©SpringerInternationalPublishingSwitzerland2017 Thisworkissubjecttocopyright.AllrightsarereservedbythePublisher,whetherthewholeorpartof the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilarmethodologynowknownorhereafterdeveloped. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publicationdoesnotimply,evenintheabsenceofaspecificstatement,thatsuchnamesareexempt fromtherelevantprotectivelawsandregulationsandthereforefreeforgeneraluse. Thepublisher,theauthorsandtheeditorsaresafetoassumethattheadviceandinformationinthis book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, express or implied, with respect to the material contained hereinorforanyerrorsoromissionsthatmayhavebeenmade. Printedonacid-freepaper ThisSpringerimprintispublishedbySpringerNature TheregisteredcompanyisSpringerInternationalPublishingAG Theregisteredcompanyaddressis:Gewerbestrasse11,6330Cham,Switzerland ’ Supervisor s Foreword I am delighted to introduce this Ph.D. dissertation thesis of my former student Dr.Yogambigai(Yoga)Velmurugu.Themeasurementsandresultspresentedhere, in the area of experimental biophysics, exemplify research of the highest quality. These results will stand out as seminal contributions that will have a far-reaching impactinthefieldofdynamicalstudiesofprotein–DNAinteractions. Thisworkaddressesafundamental probleminbiology:HowdoDNA-binding proteinsthatregulateandrepairthegenomerecognizetheir“specific”bindingsites on DNA, which are buried amidst a very large excess (billions) of “nonspecific” DNAsites?Structuralstudiesonseveralprotein–DNAcomplexeshaveshownthat DNA is often severely twisted, bent, or kinked when proteins are bound to their specific sites, indicating that sequence-dependent DNA deformability must be an importantfactorinsite-specificrecognitionmechanisms.However,ourunderstand- ing of how proteins “interrogate” potential DNA binding sites and ultimately “recognize”theirspecificsitesislacking,inlargepartbecauseoftheexperimental challenges of observing in real time the conformational changes in protein and DNAthatconferspecificity. Thebulkofsuchdynamicalstudieshavebeencarriedoutusingtechniquesthat lacked the necessary time resolution or the sensitivity. Direct observations of proteins undergoing one-dimensional (1-D) diffusion on long stretches of nonspecific DNA that mimics the “search” process indicate that proteins diffuse rapidlyonDNA,with“stepping”times(or“residence”times)perDNAsiteranging from ~50 ns to 500 μs. These studies suggest that proteins must “interrogate” potential DNA sites on submillisecond timescales, before they diffuse away, thus requiring dynamical interactions tobeinvestigatedwith adequate time resolution. Lasertemperature-jump(T-jump)spectroscopyisoneofveryfewtechniquesthat enable conformational changes in biological macromolecules to be monitored on themicro-tomillisecondstimescales,thusspanningthewindowoftimeonwhich molecularrecognitionoccurs.Inthisapproach,~10nslaserpulsesrapidlyheatupa small volume of bulk aqueous solution containing the biological samples and perturb the macromolecules of interest; time-resolved changes in the molecular v vi Supervisor’sForeword conformationsinresponsetothisT-jumpperturbationcanthenbemonitoredwith high temporal resolution using fluorescent probes that can report on changes in theseconformationswithexquisitesensitivity. TheT-jumpapproach,whenappliedtothestudyofprotein–DNAinteractions,is stillratherchallengingforaconfluenceofreasons:spectroscopicprobesneedtobe judiciously positioned without disturbing the structures of the molecules or their binding interactions; the conformational changes in protein–DNA complexes, especiallyduringtheinterrogationprocess,areoftenquitesubtle;thefluorescence signals can be at the limit of the sensitivity of the T-jump spectrometers; and increasing the concentrations of protein/DNA in the samples to improve signal intensities leads to problems with protein aggregation. These are some of the reasons why T-jump perturbation technique, although a popular approach to studyproteinandnucleicacidfolding/unfolding,wheretheconformationalchanges arelarge,hasnotbeenwidelyusedforinvestigationsofprotein–DNAinteractions. In this work, Yoga was able to overcome many of these challenges to unveil previouslyunresolveddynamicsduringsearch-interrogation-recognitionofspecific DNA sites. Below I will briefly highlight Yoga’s contributions on two systems, whichformthebulkofthiswork. The first study, in collaboration with Prof. Phoebe Rice at the University of Chicago,aimedtoelucidatetherolethatsequence-dependentDNAdynamicsand flexibility play in the mechanism of binding site recognition by Integration Host Factor (IHF), a eubacterial DNA-bending protein that binds and bends ~35 base pair long specific DNA sites into a sharp U-turn. This degree of DNA bending inducedbyaproteinisremarkable,giventhattheDNAisratherstiffontheselength scales, with a persistence length of ~150 base pairs. Previous stopped-flow and T-jumpstudies,usingFRETbetweenfluorescencelabelsattachedattheendsofa 35basepairlongcognateDNAsequence,hadshownthatIHFcanbend/kinkDNA atspecificsitesontimescalesof~10ms.Theseresultsposedapuzzle,sincethese “recognition” times were considerably slower than the 1-D diffusion times mea- sured for proteins while nonspecifically bound to DNA. Thus, the question remained: How does a rapidly diffusing protein slow down long enough at a potential binding site to enable “slow” conformational rearrangements that lead torecognition?Werethesepreviousmeasurementsmissingearlierstepsalongthe recognitionpathway? Yogafine-tunedourhome-builtT-jumpspectrometertoenableIHF-DNAbind- ing/bending kinetics to be measured with much higher sensitivity than before. These enhancements together with a new approach to analyze the kinetic traces (maximumentropyanalysis)ledtothediscoveryofafast(~100μs)DNAbending step that was sequence nonspecific. While dynamics on this timescale, which overlapped with the 1-D diffusion times of proteins “scanning” DNA, suggested that Yoga had potentially uncovered IHF interrogation dynamics, she still had to prove that these dynamics were indeed from DNA deformations and not artifacts fromdyedynamics.Thisnecessitatedcontrolmeasurementsonalargenumberof differentDNAconstructsanddifferentwaystoattachthefluorescentdyestoDNA, toruleoutcontributionsfromerroneousfluorescencesignalsunrelatedtoprotein– Supervisor’sForeword vii DNAinteractions.Yoga’spersistenceandresiliencewerecriticalindemonstrating that this rapid step was indeed from dynamics of DNA deformations that bridged the gap between rapid search and slow recognition. These studies mark the first observation, to my knowledge, of the dynamics of DNA distortions induced by a DNA-bending protein as it “probes” the DNA while searching for its site and provide us with key missing steps in the dynamical trajectory of site-specific recognition. Thesecondstudy,incollaboration withProf. Jung-HyunMinandher group at theUniversityofIllinoisatChicago,aimedtounderstandthemechanismbywhich the DNA repair protein complex Xeroderma Pigmentosum C (XPC) recognizes UV-induced and other “bulky” lesions in DNA. Structural studies on Rad4 (yeast XPC ortholog) by Min and coworkers had shown that when bound to a damaged site, Rad4 unwinds DNA and inserts an “arm” into that site to flip out (and away fromtheprotein)thedamagednucleotides.Intriguingly,theMingroupalsoshowed thatRad4,whentetheredtonormalDNA,couldalsoflipoutnormalnucleotidesto form the same “recognition” complex as with damaged DNA. These structural studies pointed to a “kinetic gating” mechanism whereby the discrimination betweennormalanddamagedDNAsites byfreelydiffusing Rad4/XPC mustrely notonthestructuraldifferencesinthethermodynamicallymoststablestatesofthe boundcomplexesbutratheronthedifferencesinthekineticsofdeformingnormal versusdamagedDNA. Yogatookuponherself thechallengetotrylaserT-jumpmeasurementsonthe Rad4-DNA complex with the goals to measure directly the kinetics of Rad4- induced DNA bending, unwinding, and nucleotide flipping, to help shed light on the damage recognition mechanism. To accomplish this, Yoga used fluorescent analogsofnucleotidesthatcanbeincorporatedwithinDNAwithoutdisturbingits structure: (1) To measure nucleotide-flipping kinetics, she incorporated 2-aminopurine (2AP), an analog of adenine, within a model DNA damaged site; (2) To measure DNA unwinding (“twisting”) dynamics, she adapted a pair of recently developed FRET probes, tC(cid:1) and tC , that are analogs of the cytosine nitro base,oneithersideofthedamagedsite.Thesecytosineanalogsarerigidlyheldin thedoublehelicalstructureofDNAvianormalWatson-Crickpairing,suchthatthe FRETefficiencybetweentheseprobes,whichisexquisitelysensitivetochangesin theirorientations,reportsdirectlyontheextentofXPC-inducedDNAunwinding. With insight gained from the high-resolution structural studies of the Min group, Yoga’skineticsexperimentsrepresentthefirstcomprehensivestudythatunveilsin unparalleleddetailsomeofthekeystepsduringDNAdamagesearchandrecogni- tion by Rad4/XPC. These measurements, which have enabled us to assemble a coarse-grainedmolecularmovieofhowDNAdamagerecognitionisaccomplished bythisprotein,willundoubtedlyhaveamajorimpactintheDNArepairaswellas molecularcancerresearchfield. In summary, this work presents state-of-the-art measurements on macromolec- ular dynamical interactions that capture site-specific recognition and that will stimulate the field of protein–DNA recognition mechanisms. Yoga’s studies have uncovered previously unresolved steps that illuminate how a searching protein viii Supervisor’sForeword interrogatesDNA deformability on timescales commensurate with the 1-D search process,andhowthisinterrogationmayleadtomomentary“stalling”oftheprotein toenableittoexecuteslowerspecificrecognitionsteps.Manyofthespectroscopic approaches used in this work, and in particular the novel application of the tC- (cid:1)-tC FRETprobesincombinationwithlaserT-jumptomeasureDNAunwind- nitro ing dynamics, are at the leading edge in the field. These approaches are also applicable to diverse systems and will likely lead to exciting new developments in the investigations of other DNA transactions such as replication, transcription, andgeneregulation. UniversityofIllinoisatChicago AnjumAnsari Chicago,IL,USA Acknowledgment Firstandforemost,IoffermydeepestgratitudetomyadvisorProf.AnjumAnsari forherexcellentguidanceandcarethroughoutineveryaspectofthisresearch.Sheis anexceptionaladvisorinallpossibleways.Shewasthereformeallthemanyyears, nevershortofalternativeideasorguidancetopickmeupwheneverIfaltered.Her patienceandconsistentsupporthelpedme overcomemany adverse situationsand completethisdissertation.TherearemanythingsIhavelearnedfromworkingwith her—about personal integrity, high expectations of excellence, and much, much more—thatextendsfarbeyondthelaboratory.NomatterwhereIgoorwhatIdo, Iwillbecarryingonthelessonswhichshetaughtmeandbebetterbecauseofit. Iwouldliketo thank the rest of mythesis committee:Prof. Mark Schlossman, Prof. Phoebe Rice, Prof. Jung-Hyun Min, Prof. Fatemeh Khalili, for all of their encouragement and insightful comments. A special thanks to Dr. Serguei Kouznetsov for teaching me the experimental technique involved in this study. His dedication and perfection helped me to learn the ability not only to just appreciatethesuccessofexperimentalendresultsbutalsotobeequallypassionate abouttheexperimentaldesignandprocedures.IsincerelythankPhysicsdepartment headProf.DavidHofmanandallmyteachersatUICfortheirhelpandencourage- mentinnumerousways. ManythanksareduetoourcollaboratorProf.PhoebeRiceattheUniversityof ChicagoforhergenerousdonationofIHFproteinanditsmutantsinlargeamounts whenever we needed it and for her help in interpreting our data. Her valuable comments, suggestions, and expertise always directed us forward in the IHF project. I also would like to thank Dr. Ying Z. Pigli at the University of Chicago forherhelpwithexpressionandpurificationofIHFproteinanditsmutants. IamevergratefultoourcollaboratorProf.Jung-HyunMinattheUICChemistry department, for her involvement in each and every step of the Rad4 project. Her thoughtfulguidance,vibrantideas,criticalcomments,andpositiveapproachwhen tackling challenges were invaluable assets and have helped me grow up as a researcher. None of the T-jump experiments would have been possible without ix
The list of books you might like
