Table Of ContentSpringer Theses
Recognizing Outstanding Ph.D. Research
Yogambigai Velmurugu
Dynamics and
Mechanism of
DNA-Bending
Proteins in Binding
Site Recognition
Springer Theses
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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
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’
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
