The human XRCC9 gene corrects chromosomal instability and mutagen sensitivities in CHO UV40 cells

Abstract

The Chinese hamster ovary (CHO) mutant UV40 cell line is hypersensitive to UV and ionizing radiation, simple alkylating agents, and DNA cross-linking agents. The mutant cells also have a high level of spontaneous chromosomal aberrations and 3-fold elevated sister chromatid exchange. We cloned and sequenced a human cDNA, designated XRCC9, that partially corrected the hypersensitivity of UV40 to mitomycin C, cisplatin, ethyl methanesulfonate, UV, and gamma-radiation. The spontaneous chromosomal aberrations in XRCC9 cDNA transformants were almost fully corrected whereas sister chromatid exchanges were unchanged. The XRCC9 genomic sequence was cloned and mapped to chromosome 9p13. The translated XRCC9 sequence of 622 amino acids has no similarity with known proteins. The 2.5-kb XRCC9 mRNA seen in the parental cells was undetectable in UV40 cells. The mRNA levels in testis were up to 10-fold higher compared with other human tissues and up to 100-fold higher compared with other baboon tissues. XRCC9 is a candidate tumor suppressor gene that might operate in a postreplication repair or a cell cycle checkpoint function.

Figure 5

Northern blots of hamster and human poly(A)⁺ RNA. Four micrograms of poly(A)⁺ RNA from each cell line was loaded per lane and probed with a 1.5-kb XRCC9 cDNA fragment (A) and subsequently with GAPDH cDNA (B). The film was exposed for 16 h or 1 h for A and B, respectively. (C) Location within the cDNA of the 1.5-kb fragment used in hybridization. The ORF is shown by the bold line.

Figure 1

Schematic diagram of cloning strategy. The primers used for amplifying the XRCC9 cDNA by PCR are indicated by arrows.

Figure 4

Differential cytotoxicity to MMC of wild-type AA8, mutant UV40, and cDNA transformant 40cXR9.31. Cells (2 × 10⁴) were inoculated in 12-well trays and incubated with MMC for 4 days (AA8) or 7 days (UV40 and 40cXR9.31) before fixation. Trays were stained with crystal violet.

Figure 2

Nucleotide sequence of XRCC9 cDNA and the translated amino acid sequence. The consensus translation initiation sequence is indicated in bold. The start and stop codons and a leucine zipper region are underlined. An in-frame stop codon (taa) upstream of the ATG start codon and the polyadenylation signal (agtaaa) upstream of the polyadenylation site are boxed.

Figure 3

Human chromosomal localization of XRCC9. (A) FISH image of a human metaphase cell showing hybridization of the genomic probe to chromosome 9p13. (B) DAPI staining of the same metaphase showing bright staining of the centromeric region of a C-group chromosome, which is diagnostic of chromosome 9.

Figure 6

Northern blots of poly(A)⁺ RNA showing relative XRCC9 mRNA expression in tissues of baboon and human. Baboon RNAs were probed with XRCC9 (A) or GAPDH (B) cDNA. Lanes: 1, brain; 2, heart; 3, kidney; 4 liver; 5, lung; 6, lymph node; 7, ovary; 8, spleen; 9, testis. Human RNAs were probed with XRCC9 (C) or actin (D) cDNA. Lanes: 1, spleen; 2, thymus; 3, prostate; 4, testis; 5, ovary; 6, small intestine; 7, colon (mucosal lining); 8, peripheral blood leukocyte. XRCC9 signals were normalized to reference RNAs as shown using cpm.