The efficiency of DNA repair in Escherichia coli ssb-1 mutant

U bakteriji Escherichia coli dva su osnovna puta popravka oštećene DNA. RecBCD put služi za popravak dvolančanih lomova. Glavni enzim ovog puta je RecBCD koji ima helikaznu, egzonukleaznu aktivnost, i sposobnost nanošenja proteina RecA na jednolančanu DNA. RecF putem popravljaju se jednolančane praznine molekule DNA. Sve navedene enzimske aktivnosti nema sadržane u jednom enzimu već ih obavljaju proteini RecQ, recJ, RecF, RecO, RecR koji su potrebni za nastanak filamenta RecA na jednolančanoj DNA. Mutantni soj recB1080 kombinira oba puta rekombinacije nadomještajući enzimske aktivnosti (nukleazna i nanošenje RecA) proteinima RecF puta. Mutacijom ssb-1 nastaje temperaturno nestabilan protein SSB-1 sa smanjenim afinitetom vezanja na jednolančanu DNA. Cilj ovog rada bio je utvrditi da li će u slučaju slabijeg vezanja proteina SSB-1 za jednolančanu DNA doći do efikasnije zamjene s proteinom RecA. Dobiveni rezultati navode na zaključak da mutacija ssb-1 inhibira aktivnost enzima RecBCD. Vrlo važnu ulogu u zamjeni tetramera SSB-1 sa proteinom RecA imaju proteini RecFOR.Damaged DNA in Escherichia coli can be repaired by two main pathways. RecBCD pathway repairs double stranded breaks. The essential enzyme this pathway uses is RecBCD which has helicase, exonuclease activity, and the capability of loading RecA protein onto single stranded DNA. RecF pathway repairs single stranded gaps made in DNA molecule. It does not have a single enzyme performing all the essential activities. RecQ, RecJ, RecF, RecO and RecR proteins are required for polimerization of RecA nucleoprotein filament on single stranded DNA. The lost activities (nuclease and RecA loading) are compensated by combining both pathways of recombination in recB1080 mutant. Mutation ssb-1 produces temperature unstable SSB-1 protein which has a lower affinity of binding to single stranded DNA. In this thesis we wanted to see if lower binding affinity of SSB-1 protein to ssDNA will result in more efficient replacement with RecA protein. Results indicate that ssb-1 mutation inhibits the activity of RecBCD enzyme. RecFOR proteins are very important for the exchange of SSB-1 tetramere with RecA protein