Rekombinacijski popravak i SOS odgovor u mutantu recA730 bakterije Escherichia coli

doctoral thesis

• Title (english): Recombinational repair and SOS response in a recA730 mutant of bacteria Escherichia coli
• Author: Ana Šimatović
• Mentor: Krunoslav Brčić-Kostić (mentor)
• Committee member: Ivana Ivančić Baće (član povjerenstva); MBZ: 217264
• Committee member: Davor Zahradka (član povjerenstva)
• Committee member: Višnja Bačun-Družina (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Biology); Zagreb
• Defense date and country: 2015, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Biology
• Universal decimal classification (UDC): 575 - General genetics. General cytogenetics

Abstract

Protein RecA730 u biokemijskim reakcijama samostalno vrši nukleaciju, a in vivo suprimira nedostatak gena recF. Određivanjem preživljenja nakon UV i gama zračenja i frekvencije konjugacijske rekombinacije utvrdili smo kako mutant suprimira istovremeni nedostatak oba faktora u nanošenju na jednolančanu DNA (RecBCD i RecFOR) te kako u mutantu recB1080 recQ može suprimirati nedostatak helikaze RecQ. Mutant ne može suprimirati istovremeni nedostatak obje egzonukleaze (RecBCD i RecJ). Za nastanak RecA filamenta koji sudjeluje u rekombinaciji i DNA popravku važno je da protein RecA veže i hidrolizira ATP. Dodatna mutacija recA730 u mutantu recA2201, koji ima fenotip mutanta recA nul jer ne hidrolizira ATP, dovodi do konformacijske promjene koja omogućuje dvostrukom mutantu recA730, 2201 da inducira SOS odgovor. Određivanjem razine SOS odgovora u bazalnim uvjetima i nakon uvedenog dvolančanog loma utvrdili smo kako je indukcija SOS odgovora u ovom dvostrukom mutantu ovisna o enzimu RecBCD te kako je za postizanje maksimalne razine SOS odgovora, koja je utvrđena u jednostrukom mutantu recA730, ipak potrebna i hidroliza vezanog ATP-a. Dodatna mutacija recA730 u mutantu recA4159, za koji se smatra da nema sposobnost vezanja ATP-a, nije omogućila indukciju SOS odgovora što upućuje da je jednostrukom mutantu recA730 za indukciju SOS odgovora potrebna konformacijska promjena do koje se dolazi vezanjem ATP-a. Istoznačni rezultati dobiveni su i u testu konjugacijske rekombinacije. Naposljetku je utvrđeno kako su vezanje i hidroliza ATP-a nužni za rekombinacijski popravak dvolančanih lomova u mutantu recA730.

Abstract (english)

The RecA730 protein is able to nucleate itself onto single stranded DNA and suppress the recombinational defect of a recF mutant. By determining cell survival after UV and gamma irradiation and conjugational recombination proficiency, we concluded that this mutant could suppress the deficiency of cells lacking RecA loading functions from both pathways (RecBCD, RecFOR), as well as the helicase deficiency in the recB1080 recQ mutant. It could not suppress the absence of exonuclease functions from both pathways (RecBCD, RecJ). RecA filament formation and its role in recombination and DNA repair depends on the RecA protein's ability to bind and hydrolyze ATP. The addition of the recA730 mutation in the recA2201 mutant, which has the phenotype of the recA null mutant since it is not able to hydrolyze ATP, results in a conformational change that enables the double mutant recA730, 2201 to induce the SOS response. By determining the basal level of the SOS response and the level after introduction of a double strand break, we concluded that the SOS response in this double mutant depends on the RecBCD enzyme and that the high level of SOS response, observed in the single mutant recA730, depends on ATP hydrolysis. The addition of the recA730 mutation in the recA4159 mutant, for which is assumed that does not bind ATP, did not restore the SOS induction in the single mutant recA730. This indicates that the SOS induction in mutant recA730 is dependent on a conformational change caused by ATP binding. Similar results were obtained in a conjugational recombination assay. The results we obtained strongly suggest that binding and hydrolyzing ATP are crucial for recombinational repair of double strand breaks in the mutant recA730.

• Keywords: Escherichia coli; homologna rekombinacija; SOS odgovor; recA730; recA730; 2201; recA730; 4159
• Keywords (english): Escherichia coli; homologous recombination; SOS response; recA730; recA730; 2201; recA730; 4159
• Language: croatian
• URN:NBN: urn:nbn:hr:217:573619
• 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: Closed access
• Created on: 2017-05-17 10:56:48