Influence of DNA heterologies on the strand exchange reaction catalyzed by RecA of Pseumonas aeruginosa

BORGOGNO MARÍA V; MONTI MARIELA R; ARGARAÑA CARLOS E

Córdoba

Congreso; XI Congreso Argentino de Microbiología General; 2015

SAMIGE

Homologous recombination (HR) plays key roles in the generation of genetic diversity by reassembling DNA sequences from total or partially homologous DNA molecules. It allows rapid acquisition of novel functions, driving adaptation and promoting speciation. However, creation of new sequence combinations by HR has to be balanced to give the highest chance of functionality maintenance of coded proteins. In bacteria, the initial stages of recombination are catalyzed by RecA, which promotes the alignment of two DNA sequences and initiates strand exchange between them. An important property of RecA is its tolerance to a limited extent of heterology which determines the efficiency of strand exchange between divergent DNA sequences and the fidelity of HR. Besides RecA, the ability to tolerate heterologies during recombination is held in check by the Mismatch Repair System (MRS). Thus, when MRS is either downregulated or inactivated the heterologies discrimination by RecA may be decisive to maintain the integrity of the genome and ultimately to avoid mutations. In the present work, we study the influence of heterologies on HR catalyzed by RecA of Pseudomonas aeruginosa. By a systematic in vitro study of oligonucleotides strand exchange using fluorescence resonance energy transfer (FRET) techniques, we examined the efficiency of RecA-catalyzed strand exchange in presence of 12 different types of single mismatches in three positions (close to the 5?, middle or 3? end) of the incoming strand, as well as the presence of small loops. Here we show that most mismatches close to the 5? end of the incoming strand had strong inhibitory effect and this was variable depending on the type of mismatch. On the other hand, most mismatches located in the middle or 3? end of the incoming strand did not show an inhibitory effect and the strand exchange efficiency was similar among different types of mismatches. Furthermore, a direct correlation between strength of inhibition and the reversibility of strand exchange reaction was observed. Therefore, RecA recognizes different types of mismatches depending on its position suggesting that differences in the intrinsic properties of DNA owing to mismatch type, neighboring nucleotide sequences and the position of the mismatch may play a role during the strand exchange. In addition, we found that the presence of small loops (+/- 1 or 2 nucleotides) diminished RecA-mediated strand exchange reaction. These results may suggest that RecA maintains a guarding role against frameshift mutations, such as it was previously described for the MRS proteins. In conclusion, RecA can effectively compare DNA segments with different types of heterologies in its search for homology.