Identification of the XPG Region That Causes the Onset of Cockayne Syndrome by Using Xpg Mutant Mice Generated by the cDNA-Mediated Knock-In Method

Abstract

In addition to xeroderma pigmentosum (XP), mutations in the human XPG gene cause early onset of Cockayne syndrome (CS) in some patients (XPG/CS). The CS-causing mutations in such patients all produce truncated XPG proteins. To test the hypothesis that the CS phenotype, with characteristics such as growth retardation and a short life span in XPG/CS patients, results from C-terminal truncations, we constructed mutants with C-terminal truncations in mouse XPG (Xpg) (from residue D811 to the stop codon [XpgD811stop] and deletion of exon 15 [Xpg⌬ex15]). In the XpgD811stop and Xpg⌬ex15 mutations, the last 360 and 183 amino acids of the protein were deleted, respectively. To generate Xpg mutant mice, we devised the shortcut knock-in method by replacing genomic DNA with a mutated cDNA fragment (cDNA-mediated knock in). The control mice, in which one-half of Xpg genomic DNA fragment was replaced with a normal Xpg cDNA fragment, had a normal growth rate, a normal life span, normal sensitivity to UV light, and normal DNA repair ability, indicating that the Xpg gene partially replaced with the normal cDNA fragment retained normal functions. The XpgD811stop homozygous mice exhibited growth retardation and a short life span, but the Xpg⌬ex15 homozygous mice did not, indicating that deletion of the last 360 amino acids results in the CS phenotype but deletion of the last 183 amino acids does not. The XpgD811stop homozygous mice, however, exhibited a slightly milder CS phenotype than did the Xpg null mutant mice, indicating that the XpgD811stop protein still retains some Xpg function that affects the severity of the CS phenotype.Xeroderma pigmentosum (XP) is a rare autosomal recessive disease clinically characterized by hypersensitivity to sunlight, abnormal pigmentation, and a predisposition to skin cancers, particularly at sun-exposed areas. XP patients are classified into eight complementation groups. Cells of patients from seven out of the eight groups (XP-A to XP-G) exhibit a defect in the early steps of the nucleotide excision repair (NER) pathway, and cells from the remaining group (XP variant) are defective in bypass DNA synthesis across DNA lesions induced by UV radiation (1,23,34).Another sun hypersensitivity disorder, Cockayne syndrome (CS), is both clinically and genetically distinct from XP and is characterized by postnatal growth failure, a short life span, and progressive neurological dysfunction, but no predisposition to cancer (29). To date, more than 140 CS cases have been reported (29) that can be classified into five complementation groups. Most patients exhibit only CS symptoms, and they belong to either group CS-A or CS-B. These symptoms arise from mutations in the CSA or CSB gene, both of which are required for the preferential removal of UV light-induced lesions in transcribed strands of genes by the transcription-coupled repair (TCR) process (14, 36).In very rare cases, complementation analyses have classified some CS patients into XP groups XP-B, XP-D, and XP-G (6,12,13,20,27,37,38). XP-B patients are ...

Tissue specific mutagenic and carcinogenic responses in NER defective mouse models

“…However, deletion of exon 15 of this gene (Xpg ex15, truncation of the last 183 amino acids) resulted in mice that develop normally. XpgD811stop homozygous mice with a C-terminal deletion of 360 amino acids exhibited growth retardation and a short life span, mimicking human CS but in a slightly milder form than the Xpg null mutant mice [86]. This indicates that the severity of the CS phenotype is influenced by the length of the truncated Xpg protein.…”

Nucleotide excision repair deficient mouse models and neurological disease

“…Deletion of exon 15 only, which is not conserved, has no effect on mice [69]. However, combining this mutation with deletion of Xpa leads to a phenotype like Xpg -/-mice [70].…”

“…Truncation of XPG in exon 11 (of 15), leading to deletion of the highly conserved C-terminus, causes a phenotype similar to, but slightly milder than, deletion of the entire gene [69]. Deletion of exon 15 only, which is not conserved, has no effect on mice [69].…”

“…Xpg E791A/E791A and Xpg D811A/D811A mice homozygous for point mutations in two amino acid residues essential for the nuclease activity of XPG develop normally [68,69]. However, the mice and cells derived from them are hypersensitive to UV.…”

XPG: Its Products and Biological Roles

“…Consistent with observations made in patients, mice in which the last 360 amino acids in the C-terminus of XPG were deleted displayed the expected symptoms associated with the XP/CS complex. 66,67 By contrast deletion of only 183 C-terminal amino acids (exon 15) resulted only in partial UV sensitivity and none of the CS characteristics. However, when this 183 amino acid deletion in XPG was combined with a deletion in XPA, it resulted in CS-like phenotypes reminiscent of XPG/CS, indicating that this deletion has serious consequences only in an NER deficient background.…”