Thesis supervisor: Professor Maia Kivisaar (University of Tartu).
Opponent: Professor Tone Tonjum, (University of Oslo, Norway).
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.