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Homologous recombination repairs potentially lethal DNA lesions such as double-strand DNA breaks (DSBs) and single-strand DNA gaps (SSGs). In Escherichia coli, DSB repair is initiated by the RecBCD enzyme that resects double-strand DNA ends and loads RecA recombinase to the emerging single-strand (ss) DNA tails. SSG repair is mediated by the RecFOR protein complex that loads RecA onto the ssDNA segment of gaped duplex. In both repair pathways, RecA catalyses reactions of homologous DNA pairing and strand exchange, while RuvABC complex and RecG helicase process recombination intermediates. In this work, we have characterised cytological changes in various recombination mutants of E. coli after three different DNA-damaging treatments: (i) expression of I-SceI endonuclease, (ii) γ-irradiation, and (iii) UV-irradiation. All three treatments caused severe chromosome segregation defects and DNA-less cell formation in the ruvABC, recG, and ruvABC recG mutants. After I-SceI expression and γ-irradiation, this phenotype was efficiently suppressed by the recB mutation, indicating that cytological defects result mostly from incomplete DSB repair. In UV-irradiated cells, the recB mutation abolished cytological defects of recG mutants and also partially suppressed the cytological defects of ruvABC recG mutants. However, neither recB nor recO mutation alone could suppress the cytological defects of UV-irradiated ruvABC mutants. The suppression was achieved only by simultaneous inactivation of the recB and recO genes. Cell survival and microscopic analysis suggest that chromosome segregation defects in UV-irradiated ruvABC mutants largely result from defective processing of stalled replication forks. The results of this study show that chromosome morphology is a valuable marker in genetic analyses of recombinational repair in E. coli.

Double strand breaks (DSBs) in E. coli chromosome (such as those induced by gamma rays) are repaired by recombination repair, during which a certain amount of DNA gets degraded. We monitored DNA degradation in gamma-irradiated cells to assess processing of DSBs. DNA degradation in irradiated cells is regulated by RecA protein concentration and its affinity of ssDNA binding, as well as by exonucleases that trim 3′-terminated ss tails. Here we determined the effects of proteins that affect formation and stability of RecA nucleofilaments on DNA degradation and cell survival. RecF and UvrD suppressed DNA degradation through RecA protein function and SOS induction, while also improving gamma survival. RecF and UvrD function in one pathway. Acting along with RecF, RecX suppressed DNA degradation and stimulated gamma-survival, which also depends on RecA protein and SOS induction. Furthermore, we determined a role in DNA degradation of several proteins that participate in DSB repair. RecN was required for DNA repair and for degradation suppression, acting on the RecABCD pathway. Furthermore, we show that SSB protein overproduction did not affect DNA degradation. Inactivation of RecG and RuvABC, proteins that catalyze the postsynaptic phase of recombination repair of DSBs, also did not affect DNA degradation, suggesting that once formed, recombination intermediates are not subject to DNA degradation, and that the postsynaptic phase is an irreversible, single-round process, unlike the presynaptic phase, which is mostly repetitive.

Genetičkom analizom ispitivana je uloga proteina RecG i RuvABC na rekombinaciju u mutantu recB1080. Rezultati su pokazali da dvostruki mutant recB1080recG ima bolje preživljenje u odnosu na recBrecG dvostruki mutant što je u skladu s činjenicom da u mutantu recB1080 postoji interakcija 2 rekombinacijska puta, RecBCD i RecFOR. Rekombinacijska sposobnost mutanta recB1080ruvABC se ne razlikuje u odnosu na mutant recBruvABC.

Provedena je genetička analiza rekombinogenosti profaga lambda u bakterijama Escherichia coli ozračenim UV svjetlom. Cilj je bio karakterizirati proces koji se događa u ozračenim bakterijama, a koji onesposobljava profag za mjesno-specifičnu rekombinaciju (s bakterijskim kromosomom) te za homolognu rekombinaciju (s inficirajućim fagom). Pokazano je da u mutantima recB2109 i recD1903, kod kojih su znatno reducirane nukleazne aktivnosti enzima RecBCD, a sačuvana njegova helikazna aktivnost, profag progresivno gubi sposobnost za mjesno-specifičnu i homolognu rekombinaciju na isti način kao i u stanicama divljeg tipa (rec+). Ispitivanja komplementacijske sposobnosti profaga pokazala su da profag koji gubi sposobnost za rekombinaciju ostaje transkripcijski aktivan, odnosno da njegov integritet ostaje sačuvan. Nadalje, križanja fag x fag su pokazala da rekombinacija izvankromosomske DNA nije inhibirana tijekom postiradijacijske inkubacije. Za razliku od mutanata recB2109 i recD1903, u "null"-mutantu recB268 rekombinogenost profaga ostaje potpuno sačuvana. Ovi rezultati pokazuju da je gubitak rekombinogenosti profaga posljedica helikazne aktivnosti enzima RecBCD. Pronađeno je također da rekombinogenost profaga ostaje sačuvana u mutantima ruvA, ruvB, ruvC i recG. (Mutacije u ovim genima oštećuju rekombinaciju na RecBCD putu.) Na temelju rezultata dobivenih u ovom radu može se zaključiti da je gubitak rekombinogenosti profaga posljedica neuspješnog rekombinacijskog popravka oštećenog bakterijskog kromosoma, koji uključuje sve enzime na RecBCD putu.