Molecular analysis of mutations in DNA polymerase η in xeroderma pigmentosum-variant patients

Abstract

Xeroderma pigmentosum variant (XP-V) cells are deficient in their ability to synthesize intact daughter DNA strands after UV irradiation. This deficiency results from mutations in the gene encoding DNA polymerase , which is required for effecting translesion synthesis (TLS) past UV photoproducts. We have developed a simple cellular procedure to identify XP-V cell strains, and have subsequently analyzed the mutations in 21 patients with XP-V. The 16 mutations that we have identified fall into three categories. Many of them result in severe truncations of the protein and are effectively null alleles. However, we have also identified five missense mutations located in the conserved catalytic domain of the protein. Extracts of cells falling into these two categories are defective in the ability to carry out TLS past sites of DNA damage. Three mutations cause truncations at the C terminus such that the catalytic domains are intact, and extracts from these cells are able to carry out TLS. From our previous work, however, we anticipate that protein in these cells will not be localized in the nucleus nor will it be relocalized into replication foci during DNA replication. The spectrum of both missense and truncating mutations is markedly skewed toward the N-terminal half of the protein. Two of the missense mutations are predicted to affect the interaction with DNA, the others are likely to disrupt the threedimensional structure of the protein. There is a wide variability in clinical features among patients, which is not obviously related to the site or type of mutation. X eroderma pigmentosum (XP) is a rare autosomal recessive disorder characterized by extreme sensitivity of the skin to sunlight-induced changes (1). In particular, affected individuals have a 1,000-fold increase in the incidence of sunlight-induced skin cancers. Genetically, XP is complex and in the majority of patients the clinical features result from a defect in one of seven genes, XPA-G, controlling nucleotide excision repair (NER). The products of these XP genes are involved in one or other of the steps in the NER process (2, 3). Approximately 20% of patients with XP are so-called XP variants (XP-V), which are defined by their normal levels of NER. Cultured fibroblasts from XP-V donors are only mildly sensitive to the killing effects of UV light, in contrast to the NER-defective XP cells, which have very marked hypersensitivity. Both NER-defective XP and XP-V cells are hypersensitive to the mutagenic effects of UV irradiation (4-6).The defect in XP-V cells is a reduced ability to replicate DNA after irradiation (also referred to as postreplication repair). XP-V cells are unable to synthesize intact daughter DNA strands on UV-irradiated templates (7). In vitro studies showed that this deficiency resulted from an inability to carry out translesion synthesis (TLS), the synthesis of DNA directly past damaged sites (8). These and other in vitro studies led to the cloning of the gene deficient in patients with XP-V (9, 10), and the demonstration that the ...

UV-B Radiation Induces Epithelial Tumors in Mice Lacking DNA Polymerase η and Mesenchymal Tumors in Mice Deficient for DNA Polymerase ι

“…This protein was expressed at reduced levels in Polh ϩ/Ϫ Poli ϩ/ϩ cells ( . In any case, this small Pol protein derived from the mutated Polh allele should be deficient in TLS activity, since it lacks an essential domain designated the polymerase-associated domain (PAD)/little finger domain/ wrist (4,37,56,61) and resembles the expected product (305 amino acids) of the mutated Polh allele of human XP2SA cells (5,74) (Fig. 2C).…”

Deep phenotyping of 89 xeroderma pigmentosum patients reveals unexpected heterogeneity dependent on the precise molecular defect

“…The clinical studies have been carried out in great detail; however, to simplify the data for the reader and to make objective comparisons between the severity of different phenotypic data, we used three severity scoring systems (described in SI Methods). For cellular analyses, UDS and UV+caffeine sensitivity for XP-V cells were carried out with cultured skin fibroblasts as described previously (11,55). Mutations in cDNA and genomic DNA were analyzed as described in SI Methods.…”

Proficient and Accurate Bypass of Persistent DNA Lesions by DinB DNA Polymerases

“…25,53 XP-V cells in culture also show increased UV-induced mutagenesis. 30 This is in striking contrast to other mammalian Y-family polymerases such as Rev1, whose action generates clear mutagenic signatures.…”

“…The regulation of TLS polymerases has clear implications for cancer, as many XP-V patients bear versions of Rad30 that are in principle competent for translesion synthesis but are lacking one or more putative regulatory domains. 53 The scope of posttranslational modification in regulating TLS is likely to be considerable.…”