Xeroderma Pigmentosum Variant Gene Research Articles

A non-catalytic role of DNA polymerase η in recruiting Rad18 and promoting PCNA monoubiquitination at stalled replication forks

Trans-lesion DNA synthesis (TLS) is a DNA damage-tolerance mechanism that uses low-fidelity DNA polymerases to replicate damaged DNA. The inherited cancer-propensity syndrome xeroderma pigmentosum variant (XPV) results from error-prone TLS of UV-damaged DNA. TLS is initiated when the Rad6/Rad18 complex monoubiquitinates proliferating cell nuclear antigen (PCNA), but the basis for recruitment of Rad18 to PCNA is not completely understood. Here, we show that Rad18 is targeted to PCNA by DNA polymerase eta (Polη), the XPV gene product that is mutated in XPV patients. The C-terminal domain of Polη binds to both Rad18 and PCNA and promotes PCNA monoubiquitination, a function unique to Polη among Y-family TLS polymerases and dissociable from its catalytic activity. Importantly, XPV cells expressing full-length catalytically-inactive Polη exhibit increased recruitment of other error-prone TLS polymerases (Polκ and Polι) after UV irradiation. These results define a novel non-catalytic role for Polη in promoting PCNA monoubiquitination and provide a new potential mechanism for mutagenesis and genome instability in XPV individuals.

DNA binding properties of human DNA polymerase η: implications for fidelity and polymerase switching of translesion synthesis

The human XPV (xeroderma pigmentosum variant) gene is responsible for the cancer-prone xeroderma pigmentosum syndrome and encodes DNA polymerase eta (pol eta), which catalyses efficient translesion synthesis past cis-syn cyclobutane thymine dimers (TT dimers) and other lesions. The fidelity of DNA synthesis by pol eta on undamaged templates is extremely low, suggesting that pol eta activity must be restricted to damaged sites on DNA. Little is known, however, about how the activity of pol eta is targeted and restricted to damaged DNA. Here we show that pol eta binds template/primer DNAs regardless of the presence of TT dimers. Rather, enhanced binding to template/primer DNAs containing TT dimers is only observed when the 3'-end of the primer is an adenosine residue situated opposite the lesion. When two nucleotides have been incorporated into the primer beyond the TT dimer position, the pol eta-template/primer DNA complex is destabilized, allowing DNA synthesis by DNA polymerases alpha or delta to resume. Our study provides mechanistic explanations for polymerase switching at TT dimer sites.

Translesion synthesis by human DNA polymerase eta across thymine glycol lesions.

The XP-V (xeroderma pigmentosum variant) gene product, human DNA polymerase eta (pol eta), catalyzes efficient and accurate translesion synthesis (TLS) past cis-syn thymine-thymine dimers (TT dimer). In addition, recent reports suggest that pol eta is involved in TLS past various other types of lesion, including an oxidative DNA damage, 8-hydroxyguanine. Here, we compare the abilities of pol alpha and pol eta to replicate across thymine glycol (Tg) using purified synthetic oligomers containing a 5R- or 5S-Tg. DNA synthesis by pol alpha was inhibited at both steps of insertion of a nucleotide opposite the lesion and extension from the resulting product, indicating that pol alpha can weakly contribute to TLS past Tg lesions. In contrast, pol eta catalyzed insertion opposite the lesion as efficient as that opposite undamaged T, while extension was inhibited especially on the 5S-Tg template. Thus, pol eta catalyzed relatively efficient TLS past 5R-Tg than 5S-Tg. To compare the TLS abilities of pol eta for these lesions, we determined the kinetic parameters of pol eta for catalyzing TLS past a TT dimer, an N-2-acetylaminofluorene-modified guanine, and an abasic site analogue. The possible mechanisms of pol eta-catalyzed TLS are discussed on the basis of these results.

DNA Replication Arrest and P53-Dependent and P53-Independent Cell Death Pathways in Xeroderma Pigmentosum Variant Cells.

INTRODUCTION. Xeroderma pigmentosum variant (XPV) cells lack the low-fidelity DNA polymerase, pol η, and as a result cannot replicate UV damaged DNA and experience prolonged arrest in the S phase (1, 2). Primary XPV fibroblasts and XPV fibroblasts transformed by HPV16(E6/E7) do not show early UV-induced apoptosis, but remain arrested for up to 48 hr without evidence of cell death (3). XPV cells transformed by SV40, however, show very early apoptosis, which is further enhanced by caffeine, and a large fraction of the cell population detaches from the substrate within 10-20 hr (3, 4). In the presence of caffeine, apoptosis is detectable in 1-2 hr. Transfection of SV40 transformed cells with the XPV gene, hRad30A, on a CMV promoter suppressed UV-induced apoptosis. Normal cells show parallel apoptotic behavior, but at a lower level. Paradoxically, when cells are assayed for colony formation after UV irradiation, the XPV cells transformed by HPV16,E6/E7) are much more sensitive than SV40 transformed or primary cells (3). Therefore the degree of apoptosis is no measure of long-term survival, and a non-apoptotic pathway of cell death, caused by DNA replication arrest, is associated with higher lethality than the apoptotic pathway.

Genomic structure, chromosomal localization and identification of mutations in the xeroderma pigmentosum variant (XPV) gene.

The xeroderma pigmentosum variant (XP-V) is one of the most common forms of this cancer-prone syndrome. XP groups A through G are characterized by defective nucleotide excision repair, whereas the XP-V phenotype is proficient in this pathway. The XPV gene encodes DNA polymerase eta, which catalyzes an accurate translesion synthesis, indicating that the XPV gene contributes tumor suppression in normal individuals. Here we describe the genomic structure and chromosomal localization of the XPV gene, which includes 11 exons covering the entire coding sequence, lacks a TATA sequence in the upstream region of the transcription-initiation, and is located at the chromosome band 6p21.1-6p12. Analyses of patient-derived XP-V cell lines strongly suggested that three of four cell lines carried homozygous mutations in the XPV gene. The fourth cell line, XP1RO, carried heterozygous point mutations in the XPV gene, one of which was located at the splice acceptor site of exon 2, resulting in the omission of exon 2 from the mature mRNA. These findings provide a basis for diagnosis and therapy of XP-V patients.

Xeroderma pigmentosum variant cells are resistant to immortalization.

Xeroderma pigmentosum (XP) is a human repair-deficient disorder that is caused by mutations in any of eight genes (A-G, V). The genes for complementation groups A-G have been cloned fully or in part, but the gene for the XP variant (XPV) has yet to be cloned. The lack of progress with XPV is in large part due to the rarity of stably transformed cell lines. We have attempted to immortalize fibroblasts from several XPV patients to obtain cell lines with which to characterize this disease and clone the appropriate gene. We have found, as have other investigators, that this XP group is very difficult to immortalize. We used a variety of approaches, including transfection with pSV ori- (a plasmid containing the simian virus (SV) 40 large T antigen) followed by spontaneous transformation, which provided stable immortal lines from Cockayne syndrome A and B, but not from XPV; transfection with pSV ori- and exposure to 3 Gy of X-rays; transfection with pSV ori-, exposure to 2 Gy of X-rays, and treatment with 1 mM ethyl methanesulfonate; transfection with human papilloma virus-16; and infection with SV40. Even though we used as many as 2 x 10(8) cells in some experiments, we were able to immortalize only one of our lines, XP30RO. Because the biochemical defect in XPV cell lines involves the capacity to replicate damaged DNA templates, perhaps the XPV gene product could be a replication factor that interacts with SV40 T antigen, and whose absence from XPV cell lines presents difficulties for the immortalization process to proceed.