New insights into host factor requirements for prokaryotic beta-recombinase-mediated reactions in mammalian cells

The prokaryotic beta -recombinase catalyzes site-specific recombination bet ween two directly oriented minimal sit sites in mammalian cells, both on ep isomic and chromatin-integrated substrates. Using a specific recombination activated gene expression system, we report the site-specific recombination activity of an enhanced green fluorescent protein (EGFP) fused version of beta -recombinase (beta -EGFP). This allows expression of active beta -reco mbinase detectable in vivo and in fixed cells by fluorescence microscopy. I n addition, cellular viability is compatible with a substantial level of ex pression of the beta -EGFP protein. Using fluorescence-activated cell sorti ng, we have been able to enrich cell populations expressing this fusion pro tein. Application of this strategy has allowed us to study in more depth th e host factor requirements for this system. Previous work showed that eukar yotic HMG1 protein was necessary and sufficient to help beta -recombinase a ctivity in vitro. The influence of ectopic expression of HMG1 protein in th e recombination process has been analyzed, indicating that HMG1 overexpress ion does not lead to a significant increase on the efficiency of beta -reco mbinase-mediated recombination both on episomal substrates and chromatin-as sociated targets. In addition, beta -recombinase-mediated recombination has been demonstrated in HMG1 deficient cells at the same levels as in wild ty pe cells. These data demonstrate the existence of cellular factors differen t from HMG-1 that can act as helpers for beta -recombinase activity in the eukaryotic environment.