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DNA Polymerase Exchange and Lesion Bypass in Escherichia Coli DNA Polymerase Exchange and Lesion Bypass in Escherichia Coli The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters Citation Kath, James Evon. 2016. DNA Polymerase Exchange and Lesion Bypass in Escherichia Coli. Doctoral dissertation, Harvard University, Graduate School of Arts & Sciences. Citable link http://nrs.harvard.edu/urn-3:HUL.InstRepos:26718716 Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of- use#LAA ! ! ! ! ! ! ! DNA!polymerase!exchange!and!lesion!bypass!in!Escherichia)coli! ! A!dissertation!presented! by! James!Evon!Kath! to! The!Committee!on!Higher!Degrees!in!Biophysics! ! in!partial!fulfillment!of!the!requirements! for!the!degree!of! Doctor!of!Philosophy! in!the!subject!of! Biophysics! ! Harvard!University! Cambridge,!Massachusetts! October!2015! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ©!2015!L!James!E.!Kath.!Some!Rights!Reserved.! ! This!work!is!licensed!under!the!Creative!Commons!Attribution!3.0!United!States!License.!To! view!a!copy!of!this!license,!visit:!http://creativecommons.org/licenses/By/3.0/us! ! ! Dissertation!Advisor:!Professor!Joseph!J.!Loparo! ! ! !!!!!!!!James!Evon!Kath! ! DNA$polymerase$exchange$and$lesion$bypass$in$Escherichia)coli$ $ Abstract$ ! Translesion! synthesis! (TLS)! alleviates! replication! stalling! at! DNA! lesions.! Bypass! of! lesions! by! specialized! translesion! DNA! polymerases! involves! exchange! with!highLfidelity!replicative!polymerases.!As!a!consequence!of!their!lesion!bypass! activity,!TLS!polymerases!are!mutagenic,!requiring!careful!regulation!of!polymerase! selection.! In! this! dissertation,! I! describe! a! singleLmolecule! reconstitution! of! polymerase! exchange! and! lesion! bypass.! Using! Escherichia) coli! polymerases! as! a! model! system,! I! have! determined! that! the! dimeric! processivity! clamp! can! simultaneously! bind! a! replicative! polymerase! and! a! translesion! polymerase,! facilitating!rapid!exchange!during!synthesis!and!lesion!bypass.!Overlapping!sets!of! polymeraseLclamp! interactions! additionally! allow! the! TLS! polymerase! Polymerase! IV! to! displace! the! replicative! polymerase! Polymerase! III.! I! finally! describe! the! observation! of! single! Polymerase! IV! molecules! in! living! cells! and! initial! efforts! to! determine!their!localization!and!dynamics!during!TLS.! $ ! ! iii! ! Contents! ! Title$page$ i$ $ Copyright$page$ ii$ $ Abstract$ iii$ $ Table$of$contents$ iv$ $ $ 1$$ Introduction$ 1$ 1.1! DNA!replication!in!Escherichia)coli!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.)))))1! 1.2! DNA!damage,!repair,!and!the!SOS!response!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.)))))3! 1.3! Translesion!synthesis!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.! !)5! 1.4! Polymerase!selection!and!exchange!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!.) )9! References) ))))!!!!!!!13! ! ! ! 2$$ Polymerase$exchange$on$single$DNA$molecules$reveals$processivity$ clamp$control$of$Polymerase$IV$translesion$synthesis$ 18$ 2.1! Introduction!!!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.)))18! 2.2! Methods:!Single!molecule!observation!of!DNA!synthesis!!.!!!!.!!!!.!!!!.)))20! ! 2.2.1! A!singleLmolecule!DNA!substrate!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!22! ! 2.2.2! A!lesionLcontaining!DNA!substrate!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!24! ! 2.2.3! Measuring!DNA!extension!by!flow!stretching!!!!.!!!!.!!!!.!!!!.!!!!.!!!!28! ! 2.2.4! SingleLmolecule!data!analysis!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!31! ! 2.2.5! Proteins!and!buffers!!!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!32! 2.3! Results!!!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!)33! ! 2.3.1! Primer!extension!by!individual!polymerases!!!!!.!!!!.!!!!.!!!!.!!!!.!! 33! ! 2.3.2! Observation!of!Pol!IIILPol!IV!exchange!and!lesion!bypass!!.! 37! ! 2.3.3! Kinetics!of!exchange!support!the!toolbelt!model!!!.!!!!.!!!!.!!!!.! 40! ! 2.3.4! Binding!of!Pol!IV!at!a!secondary!site!on!β!reduces!the!Pol!III! processivity!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!44! 2.4! Discussion!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.) 48! 2.5! Contributions!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!)52! References!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.)))).!!!!.!!!54! $ $ 3$$ Exchange$between$Escherichia)coli$Polymerases$II$and$III$on$a$single$ processivity$clamp$ 59$ 3.1! Introduction!!!.!!!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.)))59! 3.2! Methods!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!.!!!!.!!!!.!! 60))))! 3.3! Results!!!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!)61! ! 3.3.1! SingleLmolecule!analysis!of!Pol!II!synthesis!!!.!!!!.!!!!.!!!!.!!!!.!!!!.! 61! ! iv! ! ! 3.3.2! Observing!exchange!between!Pol!II!and!Pol!III!!.!!!!.!!!!.!!!!.!!!!.! 64! ! 3.3.3! Inhibition!of!Pol!III!alone!but!not!the!full!replisome!by!Pol!II! ! ! !.!!!!.!!!!.!!!!.!!!!.!!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.! 67! 3.4! Discussion!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.))70! 3.5! Contributions!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.))72! References) ))))!!!!!!!73! $ $ 4$ Observing$individual$Polymerase$IV$molecules$in$living$cells$ $ 77$ 4.1! Introduction!!!.!!!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.)))77! 4.2! Methods!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!.!!!!.!!!!.!! 79! ! 4.2.1! Strain!construction!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.! 79! ! 4.2.2! Growth!conditions!and!strain!validation!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!84! ! 4.2.3! Fluorescence!microscopy!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.! 86! ! 4.2.4! Data!analysis!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!! 88!! 4.3! Results!!!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!)89! ! 4.3.1! A!functional!Pol!IVLPAmCherry!fusion!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.! 89! ! 4.3.2! Imaging!Pol!IVLPAmCherry!molecules!with!short!exposures! ! ! ! .!!!!.!!!!.!!!!.!!!!.!!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.! 93! ! 4.3.3! Imaging!Pol!IVLPAmCherry!localizations!with!long!exposures!! ! ! .!!!!.!!!!.!!!!.!!!!.!!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.! 96! 4.4! Discussion!and!future!directions!!.!!!!.!!!!.!!!!.!!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!))101! 4.5! Contributions!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!) )102! References) ))))!!!!!!!103! $ $ 5$$ Discussion$and$future$directions$ 106$ 5.1! Conclusions!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.)))))106! 5.2! Future!directions!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.))))107! ! 5.2.1! Reconstituting!polymerase!exchange!with!the!full!replisome! ! ! ! .!!!!.!!!!.!!!!.!!!!.!!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!107! ! 5.2.2! Imaging!SSB!to!distinguish!continuous!TLS!and!lesion!skipping!! ! ! .!!!!.!!!!.!!!!.!!!!.!!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!109! ! 5.2.3! Regulators!of!polymerase!exchange!!.!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.! 110! ! 5.2.4! Observing!exchange!within!the!eukaryotic!replisome!!.!!!!.!!111!! References) ))))!!!!!!!112! $ $ Appendix$ Selecting$for$suppressors$of$Pol$IV$overproduction$lethality$$114$ A.1! Introduction!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.))114! A.2! Methods!!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.) )115! A.3! Preliminary!results!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.!!!!.! !)118! References) ))))!!!!!!!123! ! ! v! ! Acknowledgments$ $ First!and!foremost,!I!would!like!to!thank!my!advisor,!Joe!Loparo.!I!first!met! Joe!at!the!Biophysics!BBQ!in!August!2010,!when!he!was!a!new!professor!with!more! microscopes! than! lab! members.! Watching! him! establish! and! build! his! lab! was! as! important! a! training! experience! as! any! of! my! research! projects,! and! it! was! a! pleasure!to!be!a!part!of!that.!I!deeply!appreciate!Joe’s!sense!of!perspective!on!how!to! move! research! forward,! and! how! to! work! efficiently! while!
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