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. 2004 Oct 26;101(43):15410-5.
doi: 10.1073/pnas.0404587101. Epub 2004 Oct 14.

Complete absence of Cockayne syndrome group B gene product gives rise to UV-sensitive syndrome but not Cockayne syndrome

Katsuyoshi Horibata  1 , Yuka Iwamoto, Isao Kuraoka, Nicolaas G J Jaspers, Akihiro Kurimasa, Mitsuo Oshimura, Masamitsu Ichihashi, Kiyoji Tanaka
Affiliations

Complete absence of Cockayne syndrome group B gene product gives rise to UV-sensitive syndrome but not Cockayne syndrome

Katsuyoshi Horibata et al. Proc Natl Acad Sci U S A. .

Abstract

UV-sensitive syndrome (UVsS) is a rare autosomal recessive disorder characterized by photosensitivity and mild freckling but without neurological abnormalities or skin tumors. UVsS cells show UV hypersensitivity and defective transcription-coupled DNA repair of UV damage. It was suggested that UVsS does not belong to any complementation groups of known photosensitive disorders such as xeroderma pigmentosum and Cockayne syndrome (CS). To identify the gene responsible for UVsS, we performed a microcell-mediated chromosome transfer based on the functional complementation of UV hypersensitivity. We found that one of the UVsS cell lines, UVs1KO, acquired UV resistance when human chromosome 10 was transferred. Because the gene responsible for CS group B (CSB), which involves neurological abnormalities and photosensitivity as well as a defect in transcription-coupled DNA repair of UV damage, is located on chromosome 10, we sequenced the CSB gene from UVs1KO and detected a homozygous null mutation. Our results indicate that previous complementation analysis of UVs1KO was erroneous. This finding was surprising because a null mutation of the CSB gene would be expected to result in CS features such as severe developmental and neurological abnormalities. On the other hand, no mutation in the CSB cDNA and a normal amount of CSB protein was detected in Kps3, a UVsS cell line obtained from an unrelated patient, indicating genetic heterogeneity in UVsS. Possible explanations for the discrepancy in the genotype-phenotype relationship in UVs1KO are presented.

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Figures

Fig. 1.
Fig. 1.
The patient UVs1KO at ages 8 (A) and 33 (B) years.
Fig. 3.
Fig. 3.
Survival and RRS after UV irradiation in the HA-CSB/UVs1KOSV and Chr10/UVs1KOSV cells. (A) Western blot analysis of the CSB protein. WT CSB protein was absent in UVs1KOSV cells, but it was detected in normal (WI38VA13) cells, UVs1KOSV cells stably expressing HA-tagged CSB cDNA (HA-CSB/UVs1KOSV), and UVs1KOSV cells containing normal chromosome 10 (Chr10/UVs1KOSV). (B) Post-UV treatment survival of WI38VA13 cells (▪), UVs1KOSV cells (•), Chr10/UVs1KOSV cells (○), and HA-CSB/UVs1KOSV cells (▵). Chr10/UVs1KOSV and HA-CSB/UVs1KOSV cells acquired a normal level of UV sensitivity. Each point represents the mean of three independent experiments. Bars represent SE. (C) RNA synthesis ([3H]uridine incorporation) after UV irradiation in UVs1KOSV cells (•), HA-CSB/UVs1KOSV cells (▵), and WI38VA13 cells (▪). Error bars represent the SD from the mean of at least three independent experiments.
Fig. 2.
Fig. 2.
Mutations in the CSB gene in UVs1KO and CS1AN. (A) Sequence chromatograms showing mutations in UVs1KO and his parents. In UVs1KO, a C to T homozygous mutation was detected at nucleotide position 308. This C to T transition generates the nonsense codon TGA at amino acid position 77 (308CGA: 77Arg to TGA: stop). Both parents were heterozygous for this mutation. (B) RT-PCR/restriction fragment length polymorphism analysis of the C to T transition at nucleotide position 308. The site of mutation in the CSB cDNA in UVs1KO corresponds to the recognition site for the restriction enzyme BsiEI. CSB cDNA containing the mutation site was amplified by RT-PCR and digested by BsiEI. The RT-PCR products derived from HeLa cells were cut by BsiEI, generating 214- and 122-bp fragments, whereas those from UVs1KO patient were not cut, generating only a 336-bp fragment. The RT-PCR products derived from the parents generated both cut and uncut fragments, confirming that the parents were heterozygous for this mutation. (C) The predicted CSB polypeptides in patients UVs1KO and CS1AN (CS-B). CS1AN is a compound heterozygote for the nonsense mutation at amino acid residues 336 (allele a) and 857 (allele b). However, SV40-transformed CS1AN cells, CS1ANSV, expressed only allele a (14). Nuclear localization signals (gray lines) and helicase motifs (black lines) are shown.
Fig. 4.
Fig. 4.
Expression of CSB protein in UVs1KOSV, Kps3SV, and CS1ANSV cells. (A) Western blot analysis of WT and truncated CSB proteins by using an anti-CSB antibody that recognizes the N-terminal portion of a CSB polypeptide. The truncated CSB polypeptide that was predicted by the mutation in the CSB cDNA was detected in CS1ANSV cells, whereas no CSB polypeptide was detected in UVs1KOSV cells. Bands * and ** are of unknown origin but may be degraded CSB polypeptides. (B) Western blot analysis of CSB proteins by using an anti-CSB antibody that recognizes the C-terminal portion of a CSB polypeptide. Band * is a nonspecific protein. No truncated CSB polypeptides with an N-terminal deletion were detected in UVs1KOSV and CS1ANSV cells. (C) Western blot analysis of WT CSB protein by using an anti-CSB antibody that recognizes the N-terminal portion of a CSB polypeptide. As a control blot, anti-RNA polymerase II antibody was used. Note that a normal amount of WT CSB protein was detected in Kps3 cells. (D) Immunofluorescence staining of CSB proteins in WI38VA13, UVs1KOSV, and CS1ANSV cells. The CSB protein was detected with anti-CSB antibody and visualized with an Alexa Fluor 488-conjugated anti-goat IgG antibody (green). DNA was stained with TO-PRO-3 (dark red). Merged images are also shown. Note that the truncated CSB polypeptides produced in the CS1ANSV cells are retained in the nucleus.
See this image and copyright information in PMC

Comment in

  • The many faces of Cockayne syndrome.
    Spivak G. Spivak G. Proc Natl Acad Sci U S A. 2004 Oct 26;101(43):15273-4. doi: 10.1073/pnas.0406894101. Epub 2004 Oct 19. Proc Natl Acad Sci U S A. 2004. PMID: 15494443 Free PMC article. No abstract available.

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References

    1. Friedberg, E. C., Walker, G. C. & Sied, W. (1995) DNA Repair and Mutagenesis (Am. Soc. Microbiol., Washington, DC).
    1. Hanawalt, P. C. (2002) Oncogene 21, 8949-8956. - PubMed
    1. Masutani, C., Kusumoto, R., Yamada, A., Dohmae, N., Yokoi, M., Yuasa, M., Araki, M., Iwai, S., Takio, K. & Hanaoka, F. (1999) Nature 399, 700-704. - PubMed
    1. Nance, M. A. & Berry, S. A. (1992) Am. J. Med. Genet. 42, 68-84. - PubMed
    1. Mayne, L. V. & Lehmann, A. R. (1982) Cancer Res. 42, 1473-1478. - PubMed

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