The ATPase domain but not the acidic region of Cockayne syndrome group B gene product is essential for DNA repair

Abstract

Cockayne syndrome (CS) is a human genetic disorder characterized by UV sensitivity, developmental abnormalities, and premature aging. Two of the genes involved, CSA and CSB, are required for transcription-coupled repair (TCR), a subpathway of nucleotide excision repair that removes certain lesions rapidly and efficiently from the transcribed strand of active genes. CS proteins have also been implicated in the recovery of transcription after certain types of DNA damage such as those lesions induced by UV light. In this study, site-directed mutations have been introduced to the human CSB gene to investigate the functional significance of the conserved ATPase domain and of a highly acidic region of the protein. The CSB mutant alleles were tested for genetic complementation of UV-sensitive phenotypes in the human CS-B homologue of hamster UV61. In addition, the CSB mutant alleles were tested for their ability to complement the sensitivity of UV61 cells to the carcinogen 4-nitroquinoline-1-oxide (4-NQO), which introduces bulky DNA adducts repaired by global genome repair. Point mutation of a highly conserved glutamic acid residue in ATPase motif II abolished the ability of CSB protein to complement the UV-sensitive phenotypes of survival, RNA synthesis recovery, and gene-specific repair. These data indicate that the integrity of the ATPase domain is critical for CSB function in vivo. Likewise, the CSB ATPase point mutant failed to confer cellular resistance to 4-NQO, suggesting that ATP hydrolysis is required for CSB function in a TCR-independent pathway. On the contrary, a large deletion of the acidic region of CSB protein did not impair the genetic function in the processing of either UV- or 4-NQO-induced DNA damage. Thus the acidic region of CSB is likely to be dispensable for DNA repair, whereas the ATPase domain is essential for CSB function in both TCR-dependent and -independent pathways.

Figure 1

Site-directed mutations introduced in the ATPase motif II and the acidic region of CSB. The CSB protein contains the ATPase and helicase motifs conserved in superfamily 2 as well as a negatively charged domain in which 22 of 39 residues are acidic (56%). The E646Q mutation in CSB changes the highly conserved glutamic acid in ATPase motif II to glutamine (CSBE646Q). The ACMUT1 mu-tation replaces acidic residues at positions 381, 382, 384, and 385 with alanines (CSBACMUT1). The AC377 mutation deletes 10 consecutive acidic residues (378–387) from the CSB protein (CSBAC377).

Figure 2

Relative quantitative RT-PCR of CSB transcripts isolated from UV61/pc3.1-CSBwt and UV61/pc3.1-CSBE646Q. CSB expression in hamster transfectant cell lines was determined relative to the internal 18S rRNA standard as described in MATERIALS AND METHODS. RT-PCR products were electrophoresed on an 8 M urea-6% polyacrylamide gel. The RT-PCR products from the CSB mRNA and the 18S rRNA using site-specific CSB and 18S primers respectively are indicated. Reaction products from RT-PCR amplifications are as follows: lanes 1–3, UV61/pc3.1-CSBwt using CSB primers (lane 1), CSB primers plus 18S RNA primers (lane 2), and 18S RNA primers (lane 3); lanes 4–6, UV61/pc3.1-CSBE646Q using CSB primers (lane 4), CSB primers plus 18S RNA primers (lane 5), and 18S RNA primers (lane 6).

Figure 3

UV sensitivity of AA8 and isogenic clonal populations of UV61 transfectant cell lines. Cells were irradiated with UV light as described in Materials and Methods. Data (percent survival) are expressed as the number of UV-irradiated cells forming colonies as a fraction of the colonies formed by unirradiated cells and represent the average of at least three independent experiments.

Figure 4

RNA synthesis recovery of UV61 transfectant cell lines after UV irradiation with increasing dose. Cells unirradiated or UV irradiated with the indicated dose were pulse labeled with [³H]uridine 16 h after irradiation, and acid-insoluble radioactivity was determined.

Figure 5

Formation and removal of CPDs in the DHFR gene in AA8 and UV61 transfectant cell lines. Genomic DNA (10 μg) was isolated from either unirradiated (−UV) or UV irradiated (20 J/m²) cells at 0, 8, and 24 h after exposure as described in MATERIALS AND METHODS. The DNA was subsequently digested with KpnI and either treated with T4 endonuclease V (+) or untreated (−). DNA was electrophoresed through an alkaline agarose gel (0.5%) and quantitatively transferred to a nylon membrane. The membrane was hybridized to ³²P-labeled denatured double-stranded DNA probe for the DHFR gene. Hybridization of gene-specific ³²P-labeled DHFR probes to membranes was detected by PhosphorImager analysis.

Figure 6

4-NQO sensitivity of AA8 and isogenic clonal populations of UV61 transfectant cell lines. Cells were treated with 4-NQO as described in MATERIALS AND METHODS. Data (percent survival) are expressed as the number of 4-NQO-treated cells forming colonies as a fraction of the colonies formed by untreated cells and represent the average of at least three independent experiments.

Figure 7

RNA synthesis recovery of UV61 transfectant cell lines after 4-NQO treatment with increasing dose. Cells unexposed or exposed to 4-NQO at the indicated dose were pulse labeled with [³H]uridine 16 h after treatment, and acid-insoluble radioactivity was determined.