Karakterizacija kratkih zaporedij DNK, ki povzročajo kolaps replikacijskih vilic

Pavleković, Marko (author), Plavec, Janez (mentor)

DNK je ena najpomembnejših bioloških molekul, saj opravlja vlogo prenašalke dednih informacij med generacijami. Iz tega razloga je pomembno, da med podvajanjem DNK, oziroma genoma v bioloških sistemih, ne prihaja do napak. V nedavni študiji so s pomočjo inhibicije določenih regulatornih mehanizmov, ki sicer stabilizirajo replikacijske vilice in s tem skrbijo za pravilen potek podvajanja, bila odkrita mesta na DNK, na katerih pogosteje prihaja do kolapsa replikacijskih vilic. Med njimi so zelo pogosta mesta, ki vsebujejo mikrosatelitno zaporedje d(CAGAGG)n. Raziskave kažejo, da bi do kolapsa replikacijskih vilic na teh mestih lahko prišlo zaradi tvorbe sekundarnih struktur DNK, različnih od običajne B-DNK dvojne vijačnice. Strukture, ki jih tvorijo d(CAGAGG)n, še niso poznane, in so predmet nadaljnjih raziskav. V okviru diplomske naloge smo pripravili vzorce za NMR spektroskopijo v raztopini in s pomočjo le-teh naredili karakterizacijo strukture, ki jo tvori mikrosatelitno zaporedje d(CAGAGG)n. Zanimalo nas je, kakšne strukture v raztopini tvorijo krajši fragmenti mikrosatelitnega zaporedja, zato smo se osredotočili na oligonukleotida z dvema mikrosatelitnima ponovitvama: C-las (5'-d(AGGCAGAGGCAG)-3') in G-las (5'-d(AGGCAGAGGGAG)-3'). C-las v centralnem delu vsebuje eno ponovitev mikrosatelitnega zaporedja, na vsakem od koncev pa po polovico druge ponovitve (CAG na 3' in AGG na 5' koncu). Ker nas je zanimalo tudi, kako povečanje deleža purinskih baz v že tako s purinskimi bazami bogatem zaporedju vpliva na zvijanje in strukturne lastnosti, smo se osredotočili tudi na oligonukleotid G-las, v katerem smo glede na C-las uvedli substitucijo citozina v gvanin na mestu 10. S pomočjo NMR spektroskopske analize smo preučili vpliv različnih vrednosti pH in koncentracij KCl v raztopini na zvijanje in strukturo C-las in G-las. Analiza 1H NMR spektrov je pokazala, da C-las in G-las v raztopini brez dodanega KCl najverjetneje tvorita strukturo v obliki lasnice, ki vsebuje nekanonične A¬–G ali/in G–G bazne pare. V prisotnosti KCl v raztopini oligonukleotid C-las ohrani strukturo lasnice. Slednje je v nasprotju z opaženimi karakteristikami G-las, ki se po dodatku KCl zvije v alternativno strukturo, in sicer najverjetneje G-kvadrupleks, na kar sklepamo iz 1H NMR spektralnih signalov, opaženih v karakterističnem območju za G–G bazne pare povezane preko t.i. Hoogsteenovih vodikovih vezi. Nastanek alternativnih struktur G-las po dodatku KCl je časovno odvisen, kar je skladno z intermolekularnim zvitjem, in kaže, da ima substitucija C¬10>G10 in s tem povečanje deleža purinskih baz iz 10/12 na 11/12, ključen vpliv na strukturne lastnosti.

• Language: Slovenian
• Keywords: DNK, podvajanje, NMR spektroskopija
• Work type: Bachelor thesis/paper
• Typology: 2.11 - Undergraduate Thesis
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Year: 2021
• PID: 20.500.12556/RUL-129151
• COBISS.SI-ID: 75517443
• Publication date in RUL: 27.08.2021

Secondary language

• Language: English
• Title: Characterization of short DNA sequences that cause the replication fork to collapse

Abstract

DNA is one of the most important biological molecules because of its role to carry the genetic information, enabling it to be passed to the next generations. Therefore biological systems are adopted to minimize mistakes that might occur during the replication of genomic DNA. Recent studies used targeted inhibition of certain regulatory mechanisms, that under normal circumstance stabilise the replication fork enabling the replication process to make less or no mistakes and by this approach identified sites that are more often involved in replication fork collapse. Common among them are sites that include microsatellite repeats with the sequence d(CAGAGG)n. Research results indicated that the cause of replication fork collapse at these sites could be related to the formation of DNA secondary structures, that differ from the usual B-DNA double-helix. Structures formed by d(CAGAGG)n are not yet known and are subject of further research. The purpose of this thesis was to prepare samples for NMR spectroscopy in solution and furthermore perform structural study that would help to understand the folding of microsatellite repeats d(CAGAGG)n. We wanted to know what structures are formed by smaller fragments of the microsatellite sequence, therefore we focused our research on two oligonucleotides with two microsatellite repeats: C-las (5'-d(AGGCAGAGGCAG)-3') and G-las (5'-d(AGGCAGAGGGAG)-3'). The central part of C-las contains one repeat of the microsatellite sequence and half of the second repeat on each end (CAG at the 3' and AGG at the 5' end). We also wanted to investigate the structural features upon increasing purine bases content, considering that the studied DNA segment is already rich in purine residues. For this reason we studied oligonucleotide G-las, which in comparison to C-las carries C10>G10 substitution. By using 1H NMR spectroscopy we examined the effect of different pH values and concentrations of KCl in the solution on the structure of C-las and G-las. Analysis of the 1H NMR spectra showed that C-las and G-las in solution without KCl most likely form hairpin structures involving non-canonical A–G or/and G–G base pairs, or both. In the presence of KCl in solution, oligonucleotide C-las retains the form of a hairpin. On the other hand, G-las adopts alternative structures, most likely G-quadruplexes, which is consistent with 1H NMR signals observed in the spectral range characteristic for G–G base-pairs stabilized through Hoogsteen-hydrogen bonds. The time-dependent formation of these G-las structures indicates intermolecular folding topology. In addition, the differences observed for C-las and G-las demonstrate that C10>G10 substitution and thus increase of the purine base content from 10/12 to 11/12 has a profound effect on the structural properties.

• Keywords: DNA, replication, NMR spectroscopy