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doctoral thesis
Uloga enzima RecBCD i drugih nukleaza u CRISPR-Cas naivnoj adaptaciji u bakteriji Escherichia coli
Zagreb: University of Zagreb, Faculty of Science, 2021. urn:nbn:hr:217:909855
Radovčić, Marin
University of Zagreb
Faculty of Science
Department of Biology

Institutional repository: Repository of the Faculty of Science

Cite this document

Radovčić, M. (2021). Uloga enzima RecBCD i drugih nukleaza u CRISPR-Cas naivnoj adaptaciji u bakteriji Escherichia coli (Doctoral thesis). Zagreb: University of Zagreb, Faculty of Science. Retrieved from https://urn.nsk.hr/urn:nbn:hr:217:909855

Radovčić, Marin. "Uloga enzima RecBCD i drugih nukleaza u CRISPR-Cas naivnoj adaptaciji u bakteriji Escherichia coli." Doctoral thesis, University of Zagreb, Faculty of Science, 2021. https://urn.nsk.hr/urn:nbn:hr:217:909855

Radovčić, Marin. "Uloga enzima RecBCD i drugih nukleaza u CRISPR-Cas naivnoj adaptaciji u bakteriji Escherichia coli." Doctoral thesis, University of Zagreb, Faculty of Science, 2021. https://urn.nsk.hr/urn:nbn:hr:217:909855

Radovčić, M. (2021). 'Uloga enzima RecBCD i drugih nukleaza u CRISPR-Cas naivnoj adaptaciji u bakteriji Escherichia coli', Doctoral thesis, University of Zagreb, Faculty of Science, accessed 25 November 2024, https://urn.nsk.hr/urn:nbn:hr:217:909855

Radovčić M. Uloga enzima RecBCD i drugih nukleaza u CRISPR-Cas naivnoj adaptaciji u bakteriji Escherichia coli [Doctoral thesis]. Zagreb: University of Zagreb, Faculty of Science; 2021 [cited 2024 November 25] Available at: https://urn.nsk.hr/urn:nbn:hr:217:909855

M. Radovčić, "Uloga enzima RecBCD i drugih nukleaza u CRISPR-Cas naivnoj adaptaciji u bakteriji Escherichia coli", Doctoral thesis, University of Zagreb, Faculty of Science, Zagreb, 2021. Available at: https://urn.nsk.hr/urn:nbn:hr:217:909855

Metadata
TitleUloga enzima RecBCD i drugih nukleaza u CRISPR-Cas naivnoj adaptaciji u bakteriji Escherichia coli
Title (english)The role of RecBCD enzyme and other nucleases in Escherichia coli CRISPR-Cas naïve adaptation
AuthorMarin Radovčić
MentorIvana Ivančić Baće (mentor)
Committee memberDijana Škorić (predsjednik povjerenstva)
Committee memberDavor Zahradka (član povjerenstva)
Committee memberDušica Vujaklija (član povjerenstva)
GranterUniversity of Zagreb
Faculty of Science
(Department of Biology)
Zagreb
Defense date and country2021-03-01, 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
Keywords (english)
Languagecroatian
URN:NBNurn:nbn:hr:217:909855
Promotion2021
Study programmeTitle: 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 resourceText
File originBorn digital
Access conditionsOpen access
Terms of useLicence In copyright icon
Created on2021-03-16 13:29:04