The Cockayne Syndrome Group B Gene Product Is Involved in General Genome Base Excision Repair of 8-Hydroxyguanine in DNA

Abstract

Cockayne Syndrome (CS) is a human genetic disorder with two complementation groups, CS-A and CS-B. The CSB gene product is involved in transcription-coupled repair of DNA damage but may participate in other pathways of DNA metabolism. The present study investigated the role of different conserved helicase motifs of CSB in base excision repair. Stably transformed human cell lines with site-directed CSB mutations in different motifs within its putative helicase domain were established. We find that CSB null and helicase motif V and VI mutants had greater sensitivity than wild type cells to ␥-radiation. Whole cell extracts from CSB null and motif V/VI mutants had lower activity of 8-hydroxyguanine incision in DNA than wild type cells. Also, 8-hydroxyguanine accumulated more in CSB null and motif VI mutant cells than in wild type cells after exposure to ␥-radiation. We conclude that a deficiency in general genome base excision repair of selective modified DNA base(s) might contribute to CS pathogenesis. Furthermore, whereas the disruption of helicase motifs V or VI results in a CSB phenotype, mutations in other helicase motifs do not cause this effect. The biological functions of CSB in different DNA repair pathways may be mediated by distinct functional motifs of the protein.

The role of Cockayne Syndrome group B (CSB) protein in base excision repair and aging

“…The glycosylase involved specifically in the repair of this lesion has, however, not yet been identified (Jensen et al, 2003). Furthermore, cell extracts from stably transformed human cell lines with site-directed CSB mutations in various ATPase domains have shown that ATPase domains V and VI of CSB are important for the role of the protein in the processing of 8-oxoG lesions (Tuo et al, 2001), as discussed below, whereas only domain VI appears to be involved in the repair of 8-oxoA (Tuo et al, 2002b). It is possible that CSB plays an important role in the repair of oxidatively modified bases via its interaction with lesion-specific DNA glycosylases.…”

“…Importantly, after exposure to IR, CSB deficient transformed fibroblasts, mouse embryonic fibroblasts (MEF), embryonic stem (ES) cells and keratinocytes from CSB knockout mice all show a marked reduction in survival (de Waard et al, 2004;de Waard et al, 2003;Leadon and Cooper, 1993;Tuo et al, 2001). While IR induces a variety of DNA lesions including single stranded DNA breaks (SSBs), double-strand DNA breaks (DSBs) and oxidative base damage, the observed hypersensitivity has been ascribed to oxidative DNA modifications (de Waard et al, 2004;de Waard et al, 2003), which normally are repaired by BER.…”

The role of CSA in the response to oxidative DNA damage in human cells

“…The cleavage of 8-OH-Gua-containing oligonucleotides is not impaired in CS-A fibroblasts and keratinocytes CS-B cell extracts are impaired in their capacity to incise 8-OH-Gua residues present in oligonucleotides (Dianov et al, 1999;Tuo et al, 2001Tuo et al, , 2003. The capacity of cleavage of duplex oligonucleotides containing a single 8-OH-Gua by extracts from CS-A keratinocytes was tested and compared with that of extracts from normal keratinocytes (Figure 6a).…”

“…The incision activity of extracts from the CS-A cell line CS3BE was indistinguishable from that of extracts expressing wild-type CSA (Figure 6b). Extracts from the CS-B cell line CS1AN, which are defective in 8-OH-Gua cleavage (Dianov et al, 1999;Tuo et al, 2001), are shown for comparison. These findings indicate that both CSA and CSB proteins are involved in oxidative DNA damage repair, but their functions are, at least partially, different.…”

“…However, the defect of CS-B cells in oxidative damage response extends beyond the level of TCR. CS-B human and murine cells are hypersensitive to ionizing radiation (IR) and other oxidants (Tuo et al, 2001;de Waard et al, 2003) and are impaired in the repair of oxidatively induced DNA lesions (Dianov et al, 1999;Tuo et al, 2001Tuo et al, , 2003Osterod et al, 2002). Whether CSA is involved in oxidative DNA damage response is still an open question.…”