Sequestration of Mammalian Rad51-Recombination Protein into Micronuclei

Abstract

The mammalian Rad51 protein is involved in homologous recombination and in DNA damage repair. Its nuclear distribution after DNA damage is highly dynamic, and distinct foci of Rad51 protein, distributed throughout the nuclear volume, are induced within a few hours after γ irradiation; these foci then coalesce into larger clusters. Rad51-positive cells do not undergo DNA replication. Rad51 foci colocalize with both replication protein A and sites of unscheduled DNA repair synthesis and may represent a nuclear domain for recombinational DNA repair. By 24 h postirradiation, most foci are sequestered into micronuclei or assembled into Rad51-coated DNA fibers. These micronuclei and DNA fibers display genome fragmentation typical of apoptotic cell death. Other repair proteins, such as Rad52 and Gadd45, are not eliminated from the nucleus. DNA double strand breaks in repair-deficient cells or induced by the clastogen etoposide are also accompanied by the sequestering of Rad51 protein before cell death. The spindle poison colcemid causes cell cycle arrest and Rad51-foci formation without directly damaging DNA. Collectively, these observations suggest that mammalian Rad51 protein associates with damaged DNA and/or with DNA that is temporarily or irreversibly unable to replicate and these foci may subsequently be eliminated from the nucleus.

Detection of repair activity during the DNA damage-induced G2 delay in human cancer cells

“…It appears the DNA repair activity that resulted in BrdU incorporation at these sites of DNA damage may not have been su cient to result in nuclear retention of these fragments of chromatin. These observations are consistent with previous reports of the expulsion of portions of the chromatin from the nucleus following exposure to DNA damage (Haaf et al, 1999). It remains to be determined whether these may in fact represent incomplete or unsuccessful repair of DNA damage.…”

Cytokinesis-block micronucleus cytome assay

“…1-5) [3][4][5][6][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22] . This method is now also used to measure NPBs, a biomarker of dicentric chromosomes resulting from telomere end-fusions or DNA misrepair, and to measure NBUDs, a biomarker of gene amplification [20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37][38][39] . The significance of these developments and the concept of the CBMN assay as a ''cytome'' assay of chromosomal instability are explained in the following sections.…”

“…This process has been observed in cultures grown under strong selective conditions that induce gene amplification as well as under moderate folic acid deficiency [27][28][29][30][31][32][33][34][35][36][37] . Shimizu et al 31,32 showed, using in vitro experiments with mammalian cells, that amplified DNA is selectively localized to specific sites at the periphery of the nucleus and eliminated via nuclear budding to form MNi during S phase of mitosis.…”

“…The duration of the nuclear budding process, and the extrusion of the resulting MN from the cell remain largely unknown. MNi may also be formed by a budding process following exposure to g-irradiation 33 . In this process, Rad 51 recombination protein complexes are detectable throughout the entire nucleus 3 h after irradiation and then concentrate into distinct foci before being extruded from the nucleus as NBUDs.…”

DNA damage induce γ-tubulin–RAD51 nuclear complexes in mammalian cells

“…Rad51 protein relocalizes and concentrates in nuclear foci following genotoxic treatments, such as UV irradiation, IR treatments, and mitomycin C (MMC) exposure (Haaf et al, 1995). These foci are believed to be sites of repair either at DNA lesions or at broken replication forks (Tashiro et al, 1996;Haaf et al, 1999).…”