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. We first confirm that a short heterologous insert (8bp) close to the break on the recipient cleaved M13 DNA inhibits recombination with circular as well as with linear donor molecules. The results indicate that, with these substrates, recombination is initiated at the level of the break, requires uninterrupted homology on both sides of the break, and is associated with a decreasing gradient of gene conversion. When the heterologous insertion is located on the plasmid donor DNA, similar results are obtained with a circular donor DNA. In contrast, with a linear donor molecule, bearing the insert, homology requirements, in the region of the break in M13 DNA, are abolished. This last result suggests that recombination could be initiated at the extremities of the linear donor DNA.

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. Recombination with the plasmid enhances the number of plaques and also the frequency of M13 producing blue plaques. Heterologous insertions in the region surrounding the break were analyzed for their effects on initiation of recombination. The extent of repair by recombination (number of plaques) was compared with the number of blue plaques among the repaired population. Initiation of recombination is inhibited when heterologous insertions are located at 7bp from the break, on the right side as well as on the left side. A low level of recombination is measurable for 27 bp of homology but the maximum efficiency of recombination occurred with homologies of 165 or 320 bp from the break to the heterologous insertion. At 320 bp, the extent of recombinational repair remained at a plateau level but the frequency of blue plaques progressively decreases. We have also analyzed the effect of different sizes of inserts. With longer inserts, a longer length of homology adjacent to the break is required for optimum recombination. However, the size of the insert does not affect the low level of recombination that occurred with a short homology (27 bp). The results indicate that the process is initiated at or near the break, requires homology on both sides of the break and is followed by an elongation from the double strand break to the distal regions of the DNA. Our data provide some support to the double-strand-break repair model established for meiotic recombination in yeast.

Antagonistic effect of the antiestrogen 4-hydroxytamoxifen on estradiol-stimulated acetylation of nuclear high mobility group (HMG) proteins in the uterus of newborn guinea-pigs.

The effect of the antiestrogen 4-hydroxytamoxifen on the estradiol-stimulated acetylation of nuclear high mobility group (HMG) proteins was studied in the uterus of newborn (3-day-old) guinea-pig. 4-Hydroxytamoxifen (10(-6) M) selectively inhibits the stimulatory effect of estradiol (5 x 10(-8) M) on the acetylation of HMG-14 proteins 30 min after incubation with uterine tissue slices. No effect of 4-hydroxytamoxifen was observed on HMG-1 + HMG-2 or HMG-17 proteins. The data suggest that the blockage of HMG-14 acetylation is an early event in gene expression which is in relation to the antagonistic effect of the antiestrogen.

Histone acetylation decreased by estradiol in the MCF-7 human mammary cancer cell line.

The effect of estradiol (E2) on the [3H]-acetylation of nuclear histones was studied in the MCF-7 human mammary cancer cell line in culture. Cells (approximately 10(8) were incubated with 8 x 10(-6) M [3H]-acetate in the absence (control) or in the presence of estradiol (10(-5)-10(-8) M). After 20 min incubation, the nuclear histones were extracted and separated by electrophoresis, and each histone band was measured and calculated in DPM/mg protein. It was observed that only the H2 + H3 and H4 histones were associated with the [3H]-acetate. Estradiol (10(-6)-10(-8) M) provoked a significant inhibition in the incorporation of the acetate. The negative effect, in percentage of the non-treated cell value, was as follows: in E2 (10(-6) M): -25 +/- 10 (SE) for H2 + H3 and -26 +/- 5 for H4; in E2 (10(-7) M): -35 +/- 9 and -39 +/- 10; and in E2 (10(-8) M): -56 +/- 22 and -30 +/- 13 respectively. It is concluded that estradiol has a negative effect in the acetylation of H2, H3 and H4 histones of this mammary cancer cell; no acetylation or effect is observed in H1 histones. The relationship of this finding to the biological responses of the hormone is to be explored.

Estradiol enhanced acetylation of nuclear high mobility group proteins of the uterus of newborn guinea pigs.

The effect of estradiol on the acetylation of nuclear high mobility group (HMG) proteins in the uterus of newborn (3 days old) guinea pigs was studied "in vivo" and in tissue slices. In the "in vivo" studies after subcutaneous injection of 5 mCi [3H]-acetate there is a rapid (20 min) uptake of radioactive acetate in the HMG-1, HMG-2, HMG-14 and HMG-17 high mobility group proteins. In parallel studies, after administration of the same quantity of [3H]-acetate plus 20 micrograms of estradiol (E2), a selective increase in the acetylation of HMG-14 protein is observed. The preferential acetylation of HMG-14 can also be demonstrated in uterine tissue slices 20 minutes after exposure to the hormone (5 x 10(-8)M). In conclusion, the present data suggest that the acetylation of HMG proteins, in particular HMG-14, and like that of nucleosomal "core" histones, is an early event in gene activation by estradiol.