Mehanizam temperaturne osjetljivosti proteina Cas3 iz bakterije Escherichia coli

master's thesis

• Title (english): The mechanism of temperature sensitivity of the Cas3 protein in Escherichia coli
• Author: Anja Čulo
• Mentor: Ivana Ivančić Baće (mentor)
• Mentor: Petra Peharec Štefanić (mentor)
• Committee member: Ivana Ivančić Baće (predsjednik povjerenstva)
• Committee member: Petra Peharec Štefanić (član povjerenstva)
• Committee member: Martina Šeruga Musić (član povjerenstva)
• Committee member: Gordana Lacković-Venturin (član povjerenstva)
• Committee member: Sofia Ana Blažević (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Biology); Zagreb
• Defense date and country: 2017-02-24, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Biology

Abstract

Sustav CRISPR-Cas je važan mehanizam obrane prokariota od pokretnih genetičkih elementa. Sastoji se od ponavljajućih sekvenci DNA između kojih se nalaze razmaknice, koje potječu iz plazmida ili faga, te gena cas, koji su odgovorni za različite korake obrane. Transkripcijom i procesiranjem lokusa CRISPR nastaju kratke crRNA koje u kompleksu s proteinima Cas prepoznaju stranu DNA na principu komplementarnosti pri čemu nastaje R-omča - hibrid DNA i RNA i istisnutog jednog lanaca DNA. Protein Cas3 sudjeluje u zadnjoj fazi obrane, u vezanju na R-omču odakle započinje cijepati stranu DNA. Aktivnost sustava CRISPR-Cas ovisi o proteinima H-NS, koji je represor gena cas i lokusa CRISPR, HtpG, koji je neophodan za održavanje funkcionalne razine proteina Cas3, i temperaturi. Pri višoj temperaturi od 37 °C uočen je gubitak aktivnosti proteina Cas3. U ovom radu istraživan je razlog te neaktivnosti. Određivanjem sekundarne strukture pročišćenog proteina Cas3 na različitim temperaturama, uočeno je da pri temperaturi od 35 °C dolazi do konformacijske promjene u helikoidalnoj regiji proteina Cas3. Zasađivanjem faga λ potvrdili smo da je protein Cas3 izgubio aktivnost na 35 °C in vivo. Promijenjeni protein Cas3 na 37 °C potiče povećanu replikaciju plazmida tipa ColE1, neovisno o prisutnosti proteina H-NS i/ili HtpG.

Abstract (english)

The CRISPR-Cas system is a significant defense mechanism of prokaryotes against mobile genetic elements. It is composed of DNA repeats, that flank plasmid or phage derived spacer sequences, and cas genes, which are responsible for different steps of defense. Short crRNAs are transcribed from the CRISPR array. After processing, they are incorporated into protein complexes, where they guide them to the invading DNA. crRNA interference generates R-loops, RNA-DNA hybrids that contain a displaced ssDNA loop. Cas3 protein is involved in the last phase of defense in which it degrades displaced ssDNA. CRISPR-Cas system activity depends on H-NS protein, which is a repressor of cas genes and CRISPR array, HtpG protein, which is essential for maintaining functional levels of Cas3, and temperature. Incubation at 37 °C leads to reduced activity of Cas3 protein. In this work we investigated the causes of this activity reduction. By monitoring secondary structure of purified Cas3 at different temperatures, we observed a conformational change in the helical region at 35 °C. Phage sensitivity assay confirmed Cas3 activity reduction at 35 °C in vivo. Cas3 at 37 °C stimulates runaway replication of ColE1 plasmids, which does not depend on the presence of H-NS and/or HtpG.

• Keywords: CRISPR-Cas; Cas3; temperatura; H-NS; HtpG; ColE1
• Keywords (english): CRISPR-Cas; Cas3; temperature; H-NS; HtpG; ColE1
• Language: croatian
• URN:NBN: urn:nbn:hr:217:284826
• Study programme: Title: Molecular Biology; Study programme type: university; Study level: graduate; Academic / professional title: magistar/magistra molekularne biologije (magistar/magistra molekularne biologije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2017-04-20 09:04:25