Structural study of a guanine rich region in the promoter of human FANCJ helicase

Helikaza FANCJ je človeški protein, ki sodeluje pri procesu popravljanja napak DNA prek homologne rekombinacije. Mutacije helikaze ali spremembe v nivoju njenega izražanja so povezane s Fanconijevo anemijo ter rakom jajčnikov in dojk. Fanconijevo anemijo spremljajo dovzetnost za rakava obolenja in nestabilnost genoma. Helikazo FANCJ kodira gen BRIP1, ki v svoji promotorski regiji v bližini mesta začetka transkripcije vsebuje z gvanini bogato zaporedje, ki lahko tvori G-kvadruplekse. V magistrskem delu smo dokazali, da 32 nukleotidov dolgo zaporedje, ki izvira iz promotorske regije helikaze FANCJ, tvori G-kvadruplekse v raztopini. Z zamenjavami določenih nukleotidov v zaporedju smo dosegli, da oligonukleotid zavzame eno samo strukturo G-kvadrupleksa. Z eno- in dvodimenzionalnimi metodami NMR spektroskopije smo ugotovili, da oligonukleotid z dvema zamenjanima nukleotidoma tvori G-kvadrupleks s hibridno topologijo tipa 2. Z analizo talilnih krivulj smo ugotovili, da je njegova termična stabilnost podobna termični stabilnosti G-kvadrupleksa izvornega oligonukleotida brez zamenjav. Preučili smo pomembnost dolžine propelerske zanke v G-kvadrupleksu hibridne topologije tipa 2 in pokazali, da krajšanje propelerske zanke privede do spremembe strukture iz trikvartene v dvokvartetno. Ugotovili smo, da se z gvanini bogata zaporedja pojavljajo v promotorskih regijah drugih človeških helikaz, ki razvijajo G-kvadruplekse. Predpostavili smo možno biološko funkcijo sistema G-kvadrupleks – helikaza po principu povratne zanke.FANCJ helicase is a human protein, which participates in DNA repair by homologous recombination. Mutations of FANCJ or changes in its expression levels are linked to Fanconi anemia, ovarian and breast cancer. Fanconi anemia is accompanied by a higher susceptibility to cancer and genomic instability. FANCJ helicase is encoded by the BRIP1 gene, which contains a guanine rich sequence capable of forming G-quadruplexes in its promotor region, near the transcription start site. In this master’s thesis, we have confirmed that a 32 nucleotides long guanine rich sequences, which originates from the FANCJ helicase promoter region, can form G-quadruplexes in solution. With nucleotide substitutions, we have forced the oligonucleotide to adopt a single G-quadruplex structure. Using one- and two-dimensional NMR spectroscopy methods, we have determined that an oligonucleotide with two substituted nucleotides forms a G-quadruplex that adopts a hybrid type 2 topology. Using melting curve analysis, we have found out that its thermal stability is similar to the thermal stability of the parent G-quadruplex. We have studied the importance of the length of a propeller loop in the hybrid type 2 topology G-quadruplex and shown that shortening of the propeller loop leads to a change of structure from a three-quartet to a two-quartet G-quadruplex. We have observed that guanine-rich sequences are abundant in promoter regions of other human helicases, which unfold G-quadruplexes. We have also proposed a possible biological G-quadruplex – helicase system, which could work as a feedback loop