The Saccharomyces cerevisiae Ku Autoantigen Homologue Affects Radiosensitivity Only in the Absence of Homologous Recombination

In mammalian cells, all subunits of the DNA-dependent protein kinase (DNA-PK) have been implicated in the repair of DNA double-strand breaks and in V(D)J recombination. In the yeast Saccharomyces cerevisiae, we have examined the phenotype conferred by a deletion of HDF1, the putative homologue of the 70-kD subunit of the DNA-end binding Ku complex of DNA-PK. The yeast gene does not play a role in radiation-induced cell cycle checkpoint arrest in G1 and G2 or in hydroxyurea-induced checkpoint arrest in S. In cells competent for homologous recombination, we could not detect any sensitivity to ionizing radiation or to methyl methanesulfonate (MMS) conferred by a hdf1 deletion and indeed, the repair of DNA double-strand breaks was not impaired. However, if homologous recombination was disabled (rad52 mutant background), inactivation of HDF1 results in additional sensitization toward ionizing radiation and MMS. These results give further support to the notion that, in contrast to higher eukaryotic cells, homologous recombination is the favored pathway of double-strand break repair in yeast whereas other competing mechanisms such as the suggested pathway of DNA-PK-dependent direct break rejoining are only of minor importance.

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Recognition, signaling, and repair of DNA double-strand breaks produced by ionizing radiation in mammalian cells: The molecular choreography

Mutation Research/Reviews in Mutation Research ◽

10.1016/j.mrrev.2012.06.002 ◽

2012 ◽

Vol 751 (2) ◽

pp. 158-246 ◽

Cited By ~ 227

Author(s):

Larry H. Thompson

Keyword(s):

Ionizing Radiation ◽

Mammalian Cells ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

Strand Breaks

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Double-strand-break-induced homologous recombination in mammalian cells

Biochemical Society Transactions ◽

10.1042/bst0290196 ◽

2001 ◽

Vol 29 (2) ◽

pp. 196-201 ◽

Cited By ~ 186

Author(s):

R. D. Johnson ◽

M. Jasin

Keyword(s):

Homologous Recombination ◽

Sister Chromatid ◽

Mammalian Cells ◽

Indirect Evidence ◽

Strand Break ◽

Double Strand Breaks ◽

Crossing Over ◽

Dna Double Strand Breaks ◽

Dsb Repair ◽

Strand Breaks

In mammalian cells, the repair of DNA double-strand breaks (DSBs) occurs by both homologous and non-homologous mechanisms. Indirect evidence, including that from gene targeting and random integration experiments, had suggested that non-homologous mechanisms were significantly more frequent than homologous ones. However, more recent experiments indicate that homologous recombination is also a prominent DSB repair pathway. These experiments show that mammalian cells use homologous sequences located at multiple positions throughout the genome to repair a DSB. However, template preference appears to be biased, with the sister chromatid being preferred by 2–3 orders of magnitude over a homologous or heterologous chromosome. The outcome of homologous recombination in mammalian cells is predominantly gene conversion that is not associated with crossing-over. The preference for the sister chromatid and the bias against crossing-over seen in mitotic mammalian cells may have developed in order to reduce the potential for genome alterations that could occur when other homologous repair templates are utilized. In attempts to understand further the mechanism of homologous recombination, the proteins that promote this process are beginning to be identified. To date, four mammalian proteins have been demonstrated conclusively to be involved in DSB repair by homologous recombination: Rad54, XRCC2, XRCC3 and BRCAI. This paper summarizes results from a number of recent studies.

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Homologous recombination preferentially repairs heat-induced DNA double-strand breaks in mammalian cells

International Journal of Hyperthermia ◽

10.1080/02656736.2016.1252989 ◽

2016 ◽

Vol 33 (3) ◽

pp. 336-342 ◽

Cited By ~ 6

Author(s):

Akihisa Takahashi ◽

Eiichiro Mori ◽

Yosuke Nakagawa ◽

Atsuhisa Kajihara ◽

Tadaaki Kirita ◽

...

Keyword(s):

Homologous Recombination ◽

Mammalian Cells ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

Strand Breaks

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Repair of site-specific double-strand breaks in a mammalian chromosome by homologous and illegitimate recombination.

Molecular and Cellular Biology ◽

10.1128/mcb.17.1.267 ◽

1997 ◽

Vol 17 (1) ◽

pp. 267-277 ◽

Cited By ~ 176

Author(s):

R G Sargent ◽

M A Brenneman ◽

J H Wilson

Keyword(s):

Homologous Recombination ◽

Mammalian Cells ◽

Repair Process ◽

Strand Break ◽

Double Strand Breaks ◽

Illegitimate Recombination ◽

Loss Of Function ◽

Yeast Saccharomyces Cerevisiae ◽

Site Specific ◽

Strand Breaks

In mammalian cells, chromosomal double-strand breaks are efficiently repaired, yet little is known about the relative contributions of homologous recombination and illegitimate recombination in the repair process. In this study, we used a loss-of-function assay to assess the repair of double-strand breaks by homologous and illegitimate recombination. We have used a hamster cell line engineered by gene targeting to contain a tandem duplication of the native adenine phosphoribosyltransferase (APRT) gene with an I-SceI recognition site in the otherwise wild-type APRT+ copy of the gene. Site-specific double-strand breaks were induced by intracellular expression of I-SceI, a rare-cutting endonuclease from the yeast Saccharomyces cerevisiae. I-SceI cleavage stimulated homologous recombination about 100-fold; however, illegitimate recombination was stimulated more than 1,000-fold. These results suggest that illegitimate recombination is an important competing pathway with homologous recombination for chromosomal double-strand break repair in mammalian cells.

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Coordinated nuclease activities counteract Ku at single-ended DNA double-strand breaks

Nature Communications ◽

10.1038/ncomms12889 ◽

2016 ◽

Vol 7 (1) ◽

Cited By ~ 56

Author(s):

Pauline Chanut ◽

Sébastien Britton ◽

Julia Coates ◽

Stephen P. Jackson ◽

Patrick Calsou

Keyword(s):

Homologous Recombination ◽

Mammalian Cells ◽

Double Strand Breaks ◽

Exonuclease Activity ◽

Endonuclease Activity ◽

Dna Double Strand Breaks ◽

Additional Mechanism ◽

Strand Breaks ◽

Rad51 Focus ◽

Dna Resection

Abstract Repair of single-ended DNA double-strand breaks (seDSBs) by homologous recombination (HR) requires the generation of a 3′ single-strand DNA overhang by exonuclease activities in a process called DNA resection. However, it is anticipated that the highly abundant DNA end-binding protein Ku sequesters seDSBs and shields them from exonuclease activities. Despite pioneering works in yeast, it is unclear how mammalian cells counteract Ku at seDSBs to allow HR to proceed. Here we show that in human cells, ATM-dependent phosphorylation of CtIP and the epistatic and coordinated actions of MRE11 and CtIP nuclease activities are required to limit the stable loading of Ku on seDSBs. We also provide evidence for a hitherto unsuspected additional mechanism that contributes to prevent Ku accumulation at seDSBs, acting downstream of MRE11 endonuclease activity and in parallel with MRE11 exonuclease activity. Finally, we show that Ku persistence at seDSBs compromises Rad51 focus assembly but not DNA resection.

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Megabase Chromatin Domains Involved in DNA Double-Strand Breaks in Vivo

The Journal of Cell Biology ◽

10.1083/jcb.146.5.905 ◽

1999 ◽

Vol 146 (5) ◽

pp. 905-916 ◽

Cited By ~ 1606

Author(s):

Emmy P. Rogakou ◽

Chye Boon ◽

Christophe Redon ◽

William M. Bonner

Keyword(s):

Ionizing Radiation ◽

Mammalian Cells ◽

Double Strand Breaks ◽

Dna Integrity ◽

Dna Double Strand Breaks ◽

Unique Region ◽

Strand Breaks ◽

Histone H2ax ◽

Visual Confirmation

The loss of chromosomal integrity from DNA double-strand breaks introduced into mammalian cells by ionizing radiation results in the specific phosphorylation of histone H2AX on serine residue 139, yielding a specific modified form named γ-H2AX. An antibody prepared to the unique region of human γ-H2AX shows that H2AX homologues are phosphorylated not only in irradiated mammalian cells but also in irradiated cells from other species, including Xenopus laevis, Drosophila melanogaster, and Saccharomyces cerevisiae. The antibody reveals that γ-H2AX appears as discrete nuclear foci within 1 min after exposure of cells to ionizing radiation. The numbers of these foci are comparable to the numbers of induced DNA double-strand breaks. When DNA double-strand breaks are introduced into specific partial nuclear volumes of cells by means of a pulsed microbeam laser, γ-H2AX foci form at these sites. In mitotic cells from cultures exposed to nonlethal amounts of ionizing radiation, γ-H2AX foci form band-like structures on chromosome arms and on the end of broken arms. These results offer direct visual confirmation that γ-H2AX forms en masse at chromosomal sites of DNA double-strand breaks. The results further suggest the possible existence of units of higher order chromatin structure involved in monitoring DNA integrity.

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