Combating DNA damage and maintenance of genome integrity in pseudomonads
Kuupäev
2015-09-16
Autorid
Ajakirja pealkiri
Ajakirja ISSN
Köite pealkiri
Kirjastaja
Abstrakt
Efektiivne DNA replikatsioon ja DNA kahjustuste eemaldamine on erakordse tähtsusega elusrakkude genoomi terviklikkuse säilitamisel. Kõrgelt koordineeritud DNA reparatsiooni mehhanismid võimaldavad rakkudel ellu jääda ka ulatuslike rakuväliselt indutseeritud DNA kahjustuste korral, kuid on ilmselgelt olulised ka spontaanselt tekkivate DNA kahjustuste kõrvaldamisel. Nukleotiidi väljalõike reparatsioon (NER) on üks põhilisi DNA reparatsioonisüsteeme, kus UvrA, UvrB, UvrC ja UvrD valkude toimel eemaldatakse DNA kaksikahelast 12-13 nukleotiidi pikkune üksikahelaline DNA kahjustust sisaldav lõik ning seejärel sünteesib DNA polümeraas I (Pol I) selle asemele uue DNA ahela. Pol I põhifunktsiooniks rakus on DNA replikatsioonil RNA praimerite eemaldamine ja nende asemele uute DNA lõikude süntees. DNA kahjustuste korral osaleb Pol I ka reparatsioonilisel DNA sünteesil. Seega on nii NER kui ka Pol I olulised geneetilise informatsiooni säilitamiseks rakkude paljunemisel.
Doktoritöös uuriti NER, Pol I ning samuti ka DNA-d kahjustatavate ühendite rakkudest aktiivse väljaviimise osalust genoomi terviklikkuse tagamisel pseudomonaadides, mullabakteris Pseudomonas putida ning oportunistlikus inimese patogeenis P. aeruginosa.
Uuringute tulemusena selgus, et NER valkude puudumisel suureneb oluliselt homoloogilise rekombinatsiooni (HR) sagedus P. putida kromosoomis ning väheneb rakkude eluvõime. Samas tekivad NER-defektsuse korral kiiresti kompensatoorsed mutatsioonid, mis taastavad bakterite normaalse kasvu ja vähendavad HR-i sagedust. Sellist efekti NER valkude puudumisele ei ole teistes organismides varem kirjeldatud. Teiseks selgus uurimistööst, et Pol I on seni arvatust olulisem endogeenselt tekkivate reaktiivsete hapniku radikaalide tolereerimisel. Samuti ilmnes, et Pol I puudumisel osalevad DNA sünteesil spetsialiseeritud DNA polümeraasid Pol II, Pol IV ja DnaE2. Lisaks selgus, et P. aeruginosa PAO1 liini laboratoorsete tüvede puhul toimub rakke kahjustavate kemikaalide rakkudest välja viimine erineva efektiivsusega. Selline varieeruvus mõjutab bakterite tundlikkust DNA-d kahjustatavatele ühenditele ja antibiootikumidele ning raskendab oluliselt erinevate töörühmade poolt saadud tulemuste võrdlust.
Every time a living cell divides, it is confronted with a challenge to replicate its genome with high fidelity and maintain its integrity despite numerous endogenous and exogenous DNA damage. The primary strategy to avoid replication blocks is to excise the damage from the DNA strand and fill in the missing nucleotides using the intact strand as a template. In nucleotide excision repair (NER), one of the major DNA repair pathways, DNA damage is recognized and excised through a coordinated action of UvrA, UvrB, UvrC and UvrD proteins. As a next step DNA polymerase I (Pol I) fills in the resulting 12-13 nucleotide-long gap. Pol I is also involved in DNA repair synthesis in the base excision repair pathway, although the major function of Pol I is the replacement of RNA primers on the lagging strand with DNA stretches during chromosome replication. This thesis aimed to investigate the roles of NER, DNA polymerase I and the active efflux of the damaging agents in combating DNA damage in pseudomonads, a soil bacterium Pseudomonas putida and an opportunistic human pathogen P. aeruginosa. This thesis identifies that in contrast to all studied bacteria, NER deficiency results in deleterious effects on morphology, growth and viability of P. putida and enhances homologous recombination between chromosomal loci both in growing and starving cells. Furthermore, this study reveals the involvement of P. putida specialized DNA polymerases Pol II, Pol IV and DnaE2 in DNA synthesis in the absence of Pol I. This work also shows that the need of Pol I for the growth of bacteria on the rich medium is caused by the inability of the Pol I-deficient cells to cope with oxidative DNA damage more than has been thought before. Finally, this study demonstrates that the levels of active efflux extrusion of noxious compounds, one of the strategies to prevent DNA damage, vary in laboratory strains of P. aeruginosa contributing to selected resistance to various chemicals and antibiotics.
Every time a living cell divides, it is confronted with a challenge to replicate its genome with high fidelity and maintain its integrity despite numerous endogenous and exogenous DNA damage. The primary strategy to avoid replication blocks is to excise the damage from the DNA strand and fill in the missing nucleotides using the intact strand as a template. In nucleotide excision repair (NER), one of the major DNA repair pathways, DNA damage is recognized and excised through a coordinated action of UvrA, UvrB, UvrC and UvrD proteins. As a next step DNA polymerase I (Pol I) fills in the resulting 12-13 nucleotide-long gap. Pol I is also involved in DNA repair synthesis in the base excision repair pathway, although the major function of Pol I is the replacement of RNA primers on the lagging strand with DNA stretches during chromosome replication. This thesis aimed to investigate the roles of NER, DNA polymerase I and the active efflux of the damaging agents in combating DNA damage in pseudomonads, a soil bacterium Pseudomonas putida and an opportunistic human pathogen P. aeruginosa. This thesis identifies that in contrast to all studied bacteria, NER deficiency results in deleterious effects on morphology, growth and viability of P. putida and enhances homologous recombination between chromosomal loci both in growing and starving cells. Furthermore, this study reveals the involvement of P. putida specialized DNA polymerases Pol II, Pol IV and DnaE2 in DNA synthesis in the absence of Pol I. This work also shows that the need of Pol I for the growth of bacteria on the rich medium is caused by the inability of the Pol I-deficient cells to cope with oxidative DNA damage more than has been thought before. Finally, this study demonstrates that the levels of active efflux extrusion of noxious compounds, one of the strategies to prevent DNA damage, vary in laboratory strains of P. aeruginosa contributing to selected resistance to various chemicals and antibiotics.
Kirjeldus
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Märksõnad
DNA kahjustused, DNA reparatsioon, Pseudomonas, DNA damage, DNA repair