Defective DNA repair and increased genomic instability in Artemis-deficient murine cells

Abstract

In developing lymphocytes, the recombination activating gene endonuclease cleaves DNA between V, D, or J coding and recombination signal (RS) sequences to form hairpin coding and blunt RS ends, which are fused to form coding and RS joins. Nonhomologous end joining (NHEJ) factors repair DNA double strand breaks including those induced during VDJ recombination. Human radiosensitive severe combined immunodeficiency results from lack of Artemis function, an NHEJ factor with in vitro endonuclease/exonuclease activities. We inactivated Artemis in murine embryonic stem (ES) cells by targeted mutation. Artemis deficiency results in impaired VDJ coding, but not RS, end joining. In addition, Artemis-deficient ES cells are sensitive to a radiomimetic drug, but less sensitive to ionizing radiation. VDJ coding joins from Artemis-deficient ES cells, which surprisingly are distinct from the highly deleted joins consistently obtained from DNA-dependent protein kinase catalytic subunit-deficient ES cells, frequently lack deletions and often display large junctional palindromes, consistent with a hairpin coding end opening defect. Strikingly, Artemis-deficient ES cells have increased chromosomal instability including telomeric fusions. Thus, Artemis appears to be required for a subset of NHEJ reactions that require end processing. Moreover, Artemis functions as a genomic caretaker, most notably in prevention of translocations and telomeric fusions. As Artemis deficiency is compatible with human life, Artemis may also suppress genomic instability in humans.

Figure 1.

Gene targeted mutation of Artemis. (A) Schematic representation of the targeting strategy. The human Artemis cDNA and the predicted truncated transcript from the Art^N/N targeted mutation are depicted. Conserved metallo-β lactamase and β CASP (reference 78) regions are denoted and mutations identified in human RS-SCID patients (reference 39) are indicated. •, point mutations; ▿, single base deletion; bars, genomic deletions of exons. (B) Representative Southern blot analysis of ES cells. Genomic DNA from the indicated cell lines was digested with BamHI and hybridized with the 3′ probe. The germline BamHI band is 15 kb and the targeted band is 10 kb. (C) RT-PCR analysis of transcripts. RT-PCR was performed on total RNA from ES cells using control ATM primers (C lanes) and Artemis-specific primers to detect transcripts containing exons 1–4 (1 lanes) or exons 1–12 (2 lanes).

Figure 2.

IR sensitivity of Artemis-deficient ES cells. Percent survival of TC1, Art^N/N, DNA-PKcs^N/N, and XRCC4^−/− ES cells is plotted as a function of IR dose (rads). The data represents the average of three independent experiments.

Figure 3.

Sequence analysis of VDJ coding joins. Individual Cam^r/Amp^r clones were isolated and sequenced. Top boxes, junctional coding sequences flanking the left and right RSs; underlined, Potential P elements; underlined/italicized, nucleotides that cannot be unequivocally assigned to either end; center column, random insertions; parentheses, potential P element possibly formed subsequent to open and shut join with nucleotide loss; **, homology-mediated joins; right column, number of clones with the same sequence. (A) Art^N/N coding join sequences. (B) DNA-PKcs^N/N coding join sequences. (C) TC1 coding join sequences. Sequences were obtained from two independently derived Art^N/N and DNA-PKcs^N/N ES clones. Independent experiments are indicated by boxes.

Figure 4.

Chromosomal aberrations in metaphase spreads from irradiated Art^N/N ES cells. (A and B) Metaphase spreads of Art^N/N ES cells 24 h after 300 rads IR. On the left, DAPI-stained chromosomes are shown. On the right, SKY analysis is shown. Yellow arrows, chromosomal translocations; red arrow, detached centromeres and chromosomal fragments.

Figure 5.

Telomere fusions arise in Art^N/N ES cells. (A) Telomeric sequence was identified using a Cy3-labeled peptide nucleic acid probe in metaphases from Art^N/N, DNA-PKcs^N/N, and TC1 ES cells. (B) Representative short-arm telomeric fusion in an Art^N/N ES cell metaphase. Arrow represents two telomeric signals located at the point of fusion between cytogenetically invisible short arms. Adjacent, nonfused chromosomes would show four discrete signals.