Uloga enzima RecBCD i drugih nukleaza u CRISPR-Cas naivnoj adaptaciji u bakteriji Escherichia coli

doctoral thesis

• Title (english): The role of RecBCD enzyme and other nucleases in Escherichia coli CRISPR-Cas naïve adaptation
• Author: Marin Radovčić
• Mentor: Ivana Ivančić Baće (mentor)
• Committee member: Dijana Škorić (predsjednik povjerenstva)
• Committee member: Davor Zahradka (član povjerenstva)
• Committee member: Dušica Vujaklija (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Biology); Zagreb
• Defense date and country: 2021-03-01, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Biology
• Universal decimal classification (UDC): 577 - Biochemistry. Molecular biology. Biophysics

Abstract

Sustav CRISPR-Cas je obrambeni mehanizam bakterija od stranih genetičkih elemenata. Za pružanje zaštite je neophodna ugradnja fragmenta strane molekule DNA (razmaknice) u lokus CRISPR. Ugradnja nove razmaknice se naziva naivnom adaptacijom, a za ugradnju je neophodan proteinski kompleks Cas1-Cas2. Nije poznato kako nastaju fragmenti za ugradnju, no smatra se da bitnu ulogu ima nukleazna aktivnost enzima RecBCD. U ovom radu je detaljnije istražena uloga enzima RecBCD i drugih egzonukleaza u naivnoj adaptaciji. Praćenjem učinkovitosti adaptacije in vivo u sojevima E. coli mutiranim za istraživane gene je pokazano da nukleazna aktivnost enzima RecBCD nije neophodna, već samo helikazna, te da nastajanje 5'-stršećih krajeva DNA pomoću egzonukleaza pomaže adaptaciju. Utvrđeno je in vitro da kompleks Cas1-Cas2 može cijepati DNA s 5'-stršećim krajevima, a sekvenciranjem je utvrđeno da većina ugrađenih razmaknica potječe s kromosoma. Rezultati potvrđuju ulogu enzima RecBCD u stvaranju fragmenata DNA, a sam proces adaptacije ne utječe na vijabilnost.

Abstract (english)

CRISPR-Cas system is a bacterial defence mechanism against foreign genetic elements based on insertion of foreign DNA fragments (spacers) into the CRISPR array. Acquisition of new spacers is called naïve adaptation and is catalysed by Cas1-Cas2 protein complex. It is unknown how fragments for insertion are generated, but it is believed that the nuclease activity of RecBCD enzyme plays an important role. The aim of this research was to further elucidate the role of RecBCD and other exonucleases in naïve adaptation in E. coli. In vivo adaptation efficiency measurements showed that the helicase activity of RecBCD is essential and that generation of 5' overhangs by host exonucleases promotes adaptation. Cas1-Cas2 complex cleaved DNA with 5' overhangs in vitro and sequencing analysis showed that the majority of new spacers were of chromosomal origin. Overall, results confirm RecBCD's involvement in naïve adaptation with no detrimental effects on host viability.

• Keywords: E. coli; CRISPR-Cas; naivna adaptacija; Cas1-Cas2; RecBCD; rekombinacija
• Keywords (english): E. coli; CRISPR-Cas; naïve adaptation; Cas1-Cas2; RecBCD; recombination
• Language: croatian
• URN:NBN: urn:nbn:hr:217:909855
• Promotion: 2021
• Study programme: Title: Biology; Study programme type: university; Study level: postgraduate; Academic / professional title: doktor/doktorica znanosti, područje prirodnih znanosti, polje biologija (doktor/doktorica znanosti, područje prirodnih znanosti, polje biologija)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2021-03-16 13:29:04