Escherichia coli RecA protein modified with a nuclear location signal binds to chromosomes in living mammalian cells

Abstract

We tried to make a well-characterized bacterial protein function in mammalian cell nuclei. For this purpose we chose Escherichia coli RecA protein and fused its carboxy terminus to the nuclear location signal of SV40 large T-antigen by oligonucleotide-dependent modification of the gene. When injected into the cytoplasm, the modified RecA protein (T-RecA for the T-antigen signal) accumulated efficiently in the nuclei, whereas the wild-type RecA protein remained in the cytoplasm. The T-RecA protein retained its original in vivo activity, judging from the finding that uv-sensitive bacteria (recA − E. coli) became uv-resistant on transformation with the T-recA plasmid as well as the recA plasmid. For expression of the T-recA gene in mammalian cells, the 5′ region was replaced by the chicken β-actin promoter and Kozak's initiation signal. A high level of expression was observed when Chinese hamster ovary (CHO-K1) cells were transfected with this plasmid. Indirect immunofluorescence examination revealed that the T-RecA protein in nuclei of mammalian cells bound to chromatin.