Replication is one of the fundamental processes in the life cycle of the cell. The aim of replication is a duplication of genetic material. To make genetic material properly duplicated and subsequently divided into daughter cells, a large number of mechanisms of regulation of replication have been developed. One way of regulation is the proper selection and activation of replication initiation. The number and complexity of the origins of replication (ori) varies among species. They are simpler at evolution lower eukaryotes, while in higher eukaryotes are more complex. The way of recognizing these places by initiation proteins is not fully understood. Ori sequence, located downstream of the DHFR gene (dihydrofolate reductase) hamster, contains three defined origins of replication: ori β, ori β' and ori γ. Ori β DHFR sequence is strong replicator and shows activity at ectopic places. Although the sequence of ori DHFR is one of the best characterized ori sequences in metazoans and within defined functionally important regions (AT rich region, DNR region, BEND, RIP60 region, IR 4 bp), their function is still not completely understood. ORC (Origin recognition complex) is heterohexamer protein responsible for identifying origins of replication. It is assumed that several elements play a role in recognizing ori sequences of the ORC complex in metazoans. The function of the human protein Orc4 (HsOrc4) has not been fully elucidated. Our previous studies showed that HsOrc4 recognize non-canonical structures and has the ability to influence at formation of TAT triplexes and homoadenine structures. Our previous studies are done on linear fragments, and in this work we examined the function of protein HsOrc4 the circular, topologically closed DNA, (plasmid). For the analysis of the ori β DHFR, this sequence was subcloned into the plasmid which forms the structure similar to topology in vivo. In this work we analyzed the structure of functionally important regions of ori β sequence DHFR: AT rich region, DNR, BEND, RIP60. Also, we investigated binding properties of the protein HsOrc4 to supercoiled form of plasmid, and its possible role in the remodeling of plasmid topology and structure of selected regions of functional β DHFR ori sequences. We performed assays with topoisomerase and nuclease. MB nuclease (Mung Bean) recognizes and cut single stranded DNA regions, and it is used to detect non-canonical structures within the plasmid. Topoisomerase I relax plasmid and thus "lock" topology changes caused by interaction with protein HsOrc4. This research has shown that topologically closed AT rich region of ori β DHFR sequence form noncanonical structure. In the interaction with the protein HsOrc4, this structure disappears. It is possible that protein modifies this structure in direct interaction or the structural change is a consequence of changes in the the topology of the entire plasmid. Other regions studied do not form non-canonical structures at neutral pH. At acidic pH 5, DNR region occupies an alternative structure, while the AT region occupies the same structure. The survival of noncanonical structures in the AT rich regions at different pH values in the sequence of topological closed DHFR ori β, in contrast to the DNR region, suggest that the structures are not equally sensitive to pH changes. It is shown that the protein preferentially binds to HsOrc4 supercoiled form of plasmids and modify the topology of plasmids. These topology changes are reversible, since they disappear as protein is removed and for its "remembering" requires the presence of Topoisomerase I. Protein HsOrc4 intensely bind to the plasmid at pH 5 than at pH 7.5, forming large complexes. Based on the results of this study, we conclude that protein HsOrc4 introduces topological changes in plasmid that are expressed at the level of functionally important elements of ori sequences. Replikacija je jedan od osnovnih procesa u životnom ciklusu ćelije. Cilj replikacije je udvajanje genetičkog materijala. Da bi genetički materijal bio pravilno umnožen i kasnije razdvojen na ćerke ćelije, razvijen je veliki broj mehanizama regulacije replikacije. Jedan od načina regulacije je pravilan izbor i aktivacija mesta inicijacije replikacije. Broj i kompleksnost mesta inicijacije replikacije varira među vrstama. Kod evolutivno nižih eukariota su jednostavnija, dok su kod viših eukariota kompleksnija. Način prepoznavanja ovih mesta od strane inicijacionih proteina nije u potpunosti razjašnjen. Ori sekvenca, locirana nizvodno od gena DHFR (dihidrofolat reduktaza) hrčka, sadrži tri definisana ori mesta inicijacije replikacije: ori β, ori β' i ori γ. Ori β sekvenca DHFR je jak replikator i pokazuje aktivnost na ektopičnim mestima. Premda je ori sekvenca DHFR jedna od najbolje okarakterisanih ori sekvenci kod metazoa i u okviru nje definisani funkcionalno značajni regioni (AT bogati region, DNR region, BEND, RIP60 region, IR 4 bp), njihova funkcija nije u potpunosti razjašnjena. Protein ORC (eng. Origin recognition complex) je heteroheksamer odgovoran za prepoznavanje mesta inicijacije replikacije. Pretpostavka je da nekoliko elemenata ima ulogu u prepoznavanju ori sekvence od strane ORC kompleksa kod metazoa. Funkcija ljudskog proteina Orc4 (HsOrc4) do sad nije u potpunosti razjašnjena. Našim prethodnim istraživanjima, pokazali smo da HsOrc4 prepoznaje nekanonske strukture i ima sposobnost za utiče na formiranje TAT tripleksa i homoadeninskih struktura. Dosadašnja istraživanja su vršena na linearnim fragmentima, pa je u ovom radu funkcija proteina HsOrc4 ispitivana na cirkularnoj, topološki zatvorenoj DNK, odnosno plazmidu. Za analizu je odabrana sekvenca ori β DHFR, uklonirana u plazmid u kojem zauzima strukturu najsličniju in vivo topologiji. U ovom radu je analizirana struktura funkcionalno značajnih regiona ori β DHFR sekvence: AT bogatog regiona, DNR, BEND, RIP60. Ispitivano je vezivanje proteina HsOrc4 za superspiralizovanu formu plazmida i eventualna uloga u remodelovanju topologije plazmida i strukture odabranih funkcionalnih regiona ori β DHFR sekvence. Analize su vršene nukleaznim i topoizomeraznim esejom. Nukleaza MB (eng. Mung Bean) prepoznaje i iseca jednolančane DNK regione, pa je korišćena za detektovanje nekanonskih struktura unutar plazmida. Topoizomeraza I relaksira plazmid i na taj način „zaključava“ promene topologije izazvane interakcijom sa proteinom HsOrc4. Ovim istraživanjem je pokazano da topološki zatvoreni AT bogati region ori β DHFR formira nekanonsku strukturu. U interakciji sa proteinom HsOrc4, ova struktura nestaje. Moguće je da protein direktnom interakcijom modifikuje ovu strukturu ili da je promena strukture posledica izmene topologije celokupnog plazmida. Ostali ispitivani regioni ne zauzimaju nekanonske strukture na neutralnom pH. Na kiselom pH 5, DNR region zauzima alternativnu strukturu, dok AT region zauzima istu strukturu. Opstanak nekanonske strukture u okviru AT bogatog regiona na različitim pH vrednostima unutar topološki zatvorene sekvence ori β DHFR, za razliku od DNR regiona, sugeriše da strukture nisu podjednako osetljive na promene pH. Pokazano je da se protein HsOrc4 preferencijalno vezuje za superspiralizovanu formu plazmida i modifikuje topologiju plazmida. Ove promena topologije su reverzibilne, jer nestaju po uklanjanju proteina i da bi sve bile „zapamćene“, potrebno je prisustvo Topoizomeraze I. Protein HsOrc4 se intenzivnije vezuje za plazmid na pH 5 u odnosu na pH 7,5, formirajući komplekse velikih dimenzija. Na osnovu rezultata ovog istraživanja možemo zaključiti da protein HsOrc4 uvodi topološke promene u plazmidu, koje se ispoljavaju upravo na nivou funkcionalno značajnih elemenata ori sekvence.