Repair of DNA double-strand breaks (DSB) is essential for cell viability and 
genome stability. Homologous recombination repair plays an important role in DSB 
repair and impairment of this repair mechanism may lead to loss of genomic 
integrity, which is one of the hallmarks of cancer. Recent research has shown 
that the tumor suppressor genes p53 and BRCA1 and -2 are involved in the proper 
control of homologous recombination, suggesting a role of this type of repair in 
human cancer. We developed a novel assay based on recombination between two 
Green Fluorescent Protein (GFP) sequences in transiently transfected plasmid 
DNA. The plasmid construct contains an intact, emission-shifted, "blue" variant 
of GFP (BFP), with a 300 nucleotide stretch of homology to a nonfunctional copy 
of GFP. In the absence of homologous recombination only BFP is present, but 
homologous recombination can create a functional GFP. The homologous regions in 
the plasmid were constructed in both the direct and the inverted orientation of 
transcription to detect possible differences in the recombination mechanisms 
involved. A panel of human tumor cell lines was chosen on the basis of genetic 
background and chromosome integrity and tested for homologous recombination 
using this assay. The panel included cell lines with varying levels of 
karyotypic abnormalities, isogenic cell lines with normal and mutant p53, 
isogenic cell lines with or without DNA mismatch repair, BRCA1 and -2 mutant 
cell lines, and the lymphoma cell line DT40. With this assay, the observed 
differences between cell lines with the lowest and highest levels of 
recombination were about 100-fold. Increased levels of recombination were 
associated with mutant p53, whereas a low level of recombination was found in 
the BRCA1 mutant cell line. In the cell line HT1080TG, a mutagenized derivative 
of HT1080 with two mutant alleles of p53, high levels of recombination were 
found with the direct orientation but not with the inverted orientation plasmid. 
No difference in recombination was detected between two isogenic cell lines that 
only differed in DNA mismatch repair capability. We conclude that this assay can 
detect differences in homologous recombination capacity in cultured cell lines 
and that these differences follow the patterns that would be expected from the 
different genotypes of these cell lines. Future application in normal cells may 
be useful to identify genetic determinants controlling genomic integrity or to 
detect differences in DNA repair capacity in individuals.