No public access
master's thesis
Molekularna mehanika i molekularna dinamika u regulaciji metilacije DNK u ljudskim stanicama

Ivica Odorčić (2016)
University of Rijeka
Department of Biotechnology
Metadata
TitleMolecular mechanics and molecular synamics of dna methylation regulation in human cells
AuthorIvica Odorčić
Mentor(s)Željko Svedružić (thesis advisor)
Abstract
Significance: DNA methylation is a fundamental mechanism of functional organization of the human genome. The inhibitors and activators of DNA methylation can be used in tissue engineering by cellular reprogramming and in analysis of functional organization of human genome. Numerous studies have described design of inhibitors of DNA methylation but no study so far has described design of activators of DNA methylation. In this study we analyze unusual 30 fold increase in activity of human DNA methyltransferase (Dnmt1) by inosine containing DNA substrate in hope that such approach can lead us to design of novel mechanism based activators of DNA methylation Results: Using available crystal structures of Dnmt1 with hemimethylated substrates we have prepared in silico Dnmt1 in complex with hemimethylated DNA substrate that contains inosine or guanine bases at the target site. The prepared structures have been analyzed using steered molecular dynamics protocols. We find that consistent with experimental activity studies Dnmt1 just like the bacterial enzyme M.HhaI can stabilize its active site loop in closed position by hydrogen bonding between Gly1234 and the amino group on guanine base at the target site. The inosine base does not contain an amino group and it cannot support the active site loop in its closed position. Surprisingly we also find that with guanine base at the target base substrate DNA forms guanineguanine base pair that can stabilize catalytic complex and decrease the product release rates. The same complex is not stable when guanine is replaced with inosine, and therefore the product release step is much faster with an inosine base pair. Conclusions: We have found that activation of DNA methyltransferase Dnmt1 by inosine containing substrate depends on unusual guanine-guanine base pair that can be observed only with mammalian Dnmt1 but not with simpler bacterial enzymes such as M.HhaI.
Parallel title (English)Molekularna mehanika i molekularna dinamika u regulaciji metilacije DNK u ljudskim stanicama
Committee MembersŽeljko Svedružić (committee chairperson)
Roberto Antolović (committee member)
Karlo Wittine (committee member)
GranterUniversity of Rijeka
Lower level organizational unitsDepartment of Biotechnology
PlaceRijeka
StateCroatia
Scientific field, discipline, subdisciplineBIOTECHNICAL SCIENCES
Biotechnology
Study programme typeuniversity
Study levelgraduate
Study programmeMedicinal chemistry
Academic title abbreviationmag. med. chem.
Genremaster's thesis
Language English
Defense date2016-09-21
Parallel abstract (Croatian)
Značaj: Metilacija DNK je temeljni mehanizam u funkcionalnoj organizaciji ljudskog genoma. Inhibitori i aktivatori metilacije DNK mogu se koristiti za stanično reprogramiranje u regeneraciji tkiva, kao novi lijekovi, ili u bazičnim istraživanjima funkcionalne organizacije ljudskog genoma. Veliki broj studija predstavio je razvoj inhibitora metilacije DNK, međutim do sada niti jedna studija nije predstavila razvoj aktivatora metilacije DNK. U ovom radu analiziramo molekularne mehanizme koji uzrokuju do 30 puta veću aktivnost bakterijskih i ljudskih DNK metiltransferaza na DNK supstratima u kojima je baza gvanin zamijenjena bazom inozin. Rezultati: Koristeći dostupne kristalne strukture pripremili smo in silico Dnmt1 komplekse s DNK supstratima koji sadrže inozin ili gvanin na ciljnom mjestu. Razlike u silama interakcije između Dnmt1 i pripremljenih DNK supstrata analizirali smo numeričkim simulacijama navođene molekularne dinamike, te izračunom površinskih elektrostatskih polja. Simulacije su pokazale da sukladno eksperimentalnim rezultatima, Dnmt1 može slično kao i bakterijski enzim M.HhaI stabilizirati petlju aktivnog mjesta u zatvorenoj poziciji stvaranjem vodikovih veza s gvaninom u ciljnom mjestu. Slične stabilizacijske veze ne mogu se stvarati s inozinom što vodi do bržeg raspada katalitičkog kompleksa i bržeg oslobađanja produkta katalize. Nadalje, zamjena gvanina sa inozinom vodi do raspada jedinstvenog gvanin:gvanin baznog para koji stabilizira katalitički kompleks kod ljudske Dnmt1, ali ne postoji kod bakterijskog enzima M.HhaI. Brzi raspad jedinstvenog inozin:inozin kompleksa je glavni razlog zašto ljudski enzim u usporedbi s bakterijskim enzimima pokazuje i do 5 puta veću aktivaciju s DNK supstratima koji sadrže inozin na ciljnom mjestu. Zaključak: Otkrili smo da aktivacija DNK metiltransferaze Dnmt1 pomoću supstrata koji sadrži inozin ovisi o neuobičajenom gvanin:gvanin baznom paru koji je prisutan u DNK vezanoj u Dnmt1 sisavaca, ali ne i kod bakterijskih enzima poput M.HhaI.
Parallel keywords (Croatian)DNA methylation epigenetics DNA methyltransferase enzyme dynamics mechanism based activation Metilacija DNK epigenetika DNK metiltransferaze dinamika enzima aktivacija zasnovana na mehanizmu
Resource typetext
Access conditionNo public access
URN:NBNhttps://urn.nsk.hr/urn:nbn:hr:193:740167
CommitterIvana Dorotić