Characterization of two nuclear mammalian homologous DNA-pairing activities that do not require associated exonuclease activity.

We have developed an assay to study homologous DNA-pairing activities in mammalian nuclear extracts. This assay is derived from the POM blot assay, described earlier, which was specific for RecA activity in bacterial crude extracts. In the present work, proteins from mammalian nuclear extracts were resolved by electrophoresis on SDS/polyacrylamide gels and then electrotransferred onto a nitrocellulose membrane coated with circular single-stranded DNA (ssDNA).

Homologous pairing between single-stranded DNA immobilized on a nitrocellulose membrane and duplex DNA is specific for RecA activity in bacterial crude extract.

Reaction between a circular single stranded and a linear double stranded DNA molecule (ssDNA and dsDNA) provides an efficient system to study recombination mediated by RecA protein. However, classical assays using reaction in solution require highly purified enzymes. This limits biochemical studies of mutant RecA proteins from Escherichia coli or of RecA proteins from other organisms.

Structural effect of donor DNA on the initiation of recombination for double strand break repair in human nuclear extracts.

The effect of the structure of donor DNA molecules on the initiation of recombination for double strand break repair in human nuclear extracts, was investigated here. A unique double strand break was introduced into M13 duplex derivatives by digestion with restriction enzymes. After coincubation of the cleaved DNA in human nuclear extracts, with a plasmid containing M13 sequences spanning the break, double strand break repair was estimated by the plating efficiency in JM109 (RecA1) bacteria.

The DNA content of mouse two-cell embryos can be measured by microfluorimetric image analysis under conditions of cell viability.

Video-enhanced fluorescence imaging was used to quantify the DNA content in live two-cell mouse embryos. DNA was stained with the vital fluorophore Hoechst 33342. Conditions of dye concentration and irradiation were such that two-cell embryos could be kept in the constant presence of the dye for about 24 h without a major effect on their furtherin vitro viability.

Directional recombination is initiated at a double strand break in human nuclear extracts.

The involvement of a double strand break in the initiation of homologous recombination was examined in human nuclear extracts. M13 duplex derivatives, containing inserts in the LacZ' region (producing white plaques), were cleaved by restriction enzymes and coincubated in the extracts with a circular plasmid containing the LacZ' region without insert, and unable to produce plaques. Repair was estimated by the ability to produce plaques after transfection into JM109 (recA1) bacteria.