Maintenance of genome integrity is one of the crucial functions in life, to preserve the appropriate genetic information, being homologous recombination a key process in the DNA repair. I have used a novel technique, using slim-field microscopy to obtain single-molecule dynamics of two poorly described proteins, RarA and RecD2, in different recombination deficient mutants and conditions to characterize them. Single-molecule microscopy has been shown as a powerful method for in vivo characterization of proteins and its interactions. Together with genetics, I have added a new level of complexity in the regulation of homologous recombination as a multiway process in which many factors are involved in different avenues with partially overlapping functions depending on the kind of DNA damage generated.
I have characterized RarA and RecD2 as factors involved in recombination, but also in replication of the DNA, being part of both RecA-independent and RecA-dependent replication progression, and antagonistic regulators of RecA filamentation. RarA plays its role in replication through interactions with DnaB, and in recombination as a RecA positive regulator through its interactions with RecA, RecO, RecR, RecD2 and RecU. RarA is regulated by the RecQ-like helicases RecQ and RecS. RecD2 plays a role in chromosomal segregation that becomes essential in the absence of RecG or RuvAB, and is a negative regulator in homologous recombination that interacts with RecA, RarA, RecX, RecF and PcrA
