Complementation of defective translesion synthesis and UV light sensitivity in xeroderma pigmentosum variant cells by human and mouse DNA polymerase eta

Defects in the human gene XPV result in the variant form of the genetic disease xeroderma pigmentosum (XP-V). XPV encodes DNA polymerase η, a novel DNA polymerase that belongs to the UmuC/DinB/Rad30 superfamily. This polymerase catalyzes the efficient and accurate translesion synthesis of DNA past cis-syn cyclobutane di-thymine lesions. In this report we present the cDNA sequence and expression profiles of the mouse XPV gene and demonstrate its ability to complement defective DNA synthesis in XP-V cells. The mouse XPV protein shares 80.3% amino acid identity and 86.9% similarity with the human XPV protein. The recombinant mouse XPV protein corrected the inability of XP-V cell extracts to carry out DNA replication, by bypassing thymine dimers on template DNA. Transfection of the mouse or human XPV cDNA into human XP-V cells corrected UV sensitivity. Northern blot analysis revealed that the mouse XPV gene is expressed ubiquitously, but at a higher level in testis, liver, skin and thymus compared to other tissues. Although the mouse XPV gene was not induced by UV irradiation, its expression was elevated ~4-fold during cell proliferation. These results suggest that DNA polymerase η plays a role in DNA replication, though the enzyme is not essential for viability.     

Molecular analysis of ultraviolet-induced mutations in a xeroderma pigmentosum cell line

We have isolated and characterized 47 ultraviolet light-induced hprt mutants from a simian virus 40-transformed excision-repair-deficient xeroderma pigmentosum cell line (complementation group A). Twenty-one independent mutations were found, of which the majority were point mutations. Eleven of these were identified as base changes, nine of which could be attributed to ultraviolet damage on the transcribed DNA strand. Both transitions and transversions were found among the single base changes. A large proportion of the mutations (13/21) resulted in aberrant splicing of the hprt gene, suggesting that the target size for mutations resulting in aberrant splicing must be quite large. A small number of spontaneous mutations were identified, most of which were large deletions. Our data provide a spectrum for the intrinsic mutations resulting from ultraviolet damage in human cells in the absence of repair.     

DNA polymerase eta, the product of the xeroderma pigmentosum variant gene and a target of p53, modulates the DNA damage checkpoint and p53 activation

DNA polymerase eta (PolH) is the product of the xeroderma pigmentosum variant (XPV) gene and a well-characterized Y-family DNA polymerase for translesion synthesis. Cells derived from XPV patients are unable to faithfully bypass UV photoproducts and DNA adducts and thus acquire genetic mutations. Here, we found that PolH can be up-regulated by DNA breaks induced by ionizing radiation or chemotherapeutic agents, and knockdown of PolH gives cells resistance to apoptosis induced by DNA breaks in multiple cell lines and cell types in a p53-dependent manner. To explore the underlying mechanism, we examined p53 activation upon DNA breaks and found that p53 activation is impaired in PolH knockdown cells and PolH-null primary fibroblasts. Importantly, reconstitution of PolH into PolH knockdown cells restores p53 activation. Moreover, we provide evidence that, upon DNA breaks, PolH is partially colocalized with phosphorylated ATM at gamma-H2AX foci and knockdown of PolH impairs ATM to phosphorylate Chk2 and p53. However, upon DNA damage by UV, PolH knockdown cells exhibit two opposing temporal responses: at the early stage, knockdown of PolH suppresses p53 activation and gives cells resistance to UV-induced apoptosis in a p53-dependent manner; at the late stage, knockdown of PolH suppresses DNA repair, leading to sustained activation of p53 and increased susceptibility to apoptosis in both a p53-dependent and a p53-independent manner. Taken together, we found that PolH has a novel role in the DNA damage checkpoint and that a p53 target can modulate the DNA damage response and subsequently regulate p53 activation.     

Reevaluation of the role of DNA polymerase theta in somatic hypermutation of immunoglobulin genes

DNA polymerase theta has been implicated in the process of somatic hypermutation in immunoglobulin variable genes based on several reports of alterations in the frequency and spectra of mutations from Polq(-/-) mice. However, these studies have contrasting results on mutation frequencies and the types of nucleotide substitutions, which question the role of polymerase theta in hypermutation. DNA polymerase eta has a dominant effect on mutation and may substitute in the absence of polymerase theta to affect the pattern. Therefore, we have examined mutation in mice deficient for both polymerases theta and eta. The mutation frequencies in rearranged variable genes from Peyer's patches were similar in wild type, Polq(-/-), Polh(-/-), and Polq(-/-)Polh(-/-) mice. The types of substitutions were also similar between wild type and Polq(-/-) clones, and between Polh(-/-) and Polq(-/-)Polh(-/-) clones. Furthermore, there was no difference in heavy chain class switching in splenic B cells from the four groups of mice. These results indicate that polymerase theta does not play a significant role in the generation of somatic mutation in immunoglobulin genes.     

Replication of damaged DNA: molecular defect in xeroderma pigmentosum variant cells.

Individuals with Xeroderma pigmentosum (XP) syndrome have a genetic predisposition to sunlight-induced skin cancer. Genetically different forms of XP have been identified by cell fusion. Cells of individuals expressing the classical form of XP (complementation groups A through G) are deficient in the nucleotide excision repair (NER) pathway. In contrast, the cells belonging to the variant class of XP (XPV) are NER-proficient and are only slightly more sensitive than normal cells to the killing action of UV light radiation. The XPV fibroblasts replicate damaged DNA generating abnormally short fragments either in vivo [A.R. Lehmann, The relationship between pyramidine dimers and replicating DNA in UV-irradiated human fibroblasts, Nucleic Acids Res. 7 (1979) 1901-1912; S.D. Park, J.E. Cleaver, Postreplication repair: question of its definition and possible alteration in Xeroderma pigmentosum cell strains, Proc. Natl. Acad. Sci. U.S.A. 76 (1979) 3927-3931.] or in vitro [S.M. Cordeiro, L.S. Zaritskaya, L.K. Price, W.K. Kaufmann, Replication fork bypass of a pyramidine dimer blocking leading strand DNA synthesis, J. Biol. Chem. 272 (1997) 13945-13954; D.L. Svoboda, L.P. Briley, J.M. Vos, Defective bypass replication of a leading strand cyclobutane thymine dimer in Xeroderma pigmentosum variant cell extracts, Cancer Res. 58 (1998) 2445-2448; I. Ensch-Simon, P.M. Burgers, J.S. Taylor, Bypass of a site-specific cis-syn thymine dimer in an SV40 vector during in vitro replication by HeLa and XPV cell-free extracts, Biochemistry 37 (1998) 8218-8226.], suggesting that in XPV cells, replication has an increased probability of being blocked at a lesion. Furthermore, extracts from XPV cells were found to be defective in translesion synthesis [A. Cordonnier, A.R. Lehmann, R.P.P. Fuchs, Impaired translesion synthesis in Xeroderma pigmentosum variant extracts, Mol. Cell. Biol. 19 (1999) 2206-2211.]. Recently, Masutani et al. [C. Masutani, M. Araki, A. Yamada, R. Kusomoto, T. Nogimori, T. Maekawa, S. Iwai, F. Hanaoka, Xeroderma pigmentosum variant (XP-V) correcting protein from HeLa cells has a thymine dimer bypass DNA polymerase activity, EMBO J. 18 (1999) 3491-3501.] have shown that the XPV defect can be corrected by a novel human DNA polymerase, homologue to the yeast DNA polymerase eta, which is able to replicate past cyclobutane pyrimidine dimers in DNA templates. This review focuses on our current understanding of translesion synthesis in mammalian cells whose defect, unexpectedly, is responsible for the hypermutability of XPV cells and for the XPV pathology.     

The xeroderma pigmentosum pathway: decision tree analysis of DNA quality

The nucleotide excision repair (NER) system is a fundamental cellular stress response that uses only a handful of DNA binding factors, mutated in the cancer-prone syndrome xeroderma pigmentosum (XP), to detect an astounding diversity of bulky base lesions, including those induced by ultraviolet light, electrophilic chemicals, oxygen radicals and further genetic insults. Several of these XP proteins are characterized by a mediocre preference for damaged substrates over the native double helix but, intriguingly, none of them recognizes injured bases with sufficient selectivity to account for the very high precision of bulky lesion excision. Instead, substrate versatility as well as damage specificity and strand selectivity are achieved by a multistage quality control strategy whereby different subunits of the XP pathway, in succession, interrogate the DNA double helix for a distinct abnormality in its structural or dynamic parameters. Through this step-by-step filtering procedure, the XP proteins operate like a systematic decision making tool, generally known as decision tree analysis, to sort out rare damaged bases embedded in a vast excess of native DNA. The present review is focused on the mechanisms by which multiple XP subunits of the NER pathway contribute to the proposed decision tree analysis of DNA quality in eukaryotic cells.     

The role of DNA polymerase eta in UV mutational spectra

UV irradiation generates predominantly cyclobutane pyrimidine dimers (CPDs) and (6-4) photoproducts in DNA. CPDs are thought to be responsible for most of the UV-induced mutations. Thymine-thymine CPDs, and probably also CPDs containing cytosine, are replicated in vivo in a largely accurate manner by a DNA polymerase eta (Pol eta) dependent process. Pol eta is encoded by the POLH (XPV) gene in humans. In order to clarify the specific role of Pol eta in UV mutagenesis, we have used an siRNA knockdown approach in combination with a supF shuttle vector which replicates in mammalian cells. This strategy provides an advantage over studying mutagenesis in cell lines derived from normal individuals and XP-V patients, since the genetic background of the cells is identical. Synthetic RNA duplexes were used to inhibit Pol eta expression in 293T cells. The reduction of Pol eta mRNA and protein was greater than 90%. The supF shuttle vector was irradiated with UVC and replicated in 293T cells in presence of anti-Pol eta siRNA. The supF mutant frequency was increased by up to 3.6-fold in the siRNA knockdown cells relative to control cells confirming that Pol eta plays an important role in mutation avoidance and that the pol eta knockdown was efficient. UV-induced supF mutants were sequenced from siRNA-treated cells and controls. Surprisingly, neither the type of mutations nor their distribution along the supF gene were substantially different between controls and siRNA knockdown cells and were predominantly C to T and CC to TT transitions at dipyrimidine sites. The data are compatible with two models. (i) Incorrect replication of cytosine-containing photoproducts by a polymerase other than Pol eta produces similar mutations as when Pol eta is present but at a higher frequency. (ii) Due to lack of Pol eta or low levels of remaining Pol eta, lesion replication is delayed allowing more time for cytosine deamination within CPDs to occur. We provide proof of principle that siRNA technology can be used to dissect the in vivo roles of lesion bypass DNA polymerases in DNA damage-induced mutagenesis.     

Similar distributions of repaired sites in chromatin of normal and xeroderma pigmentosum variant cells damaged by ultraviolet light

Excision repair of damage from ultraviolet light in both normal and xeroderma pigmentosum variant fibroblasts at early times after irradiation occurred preferentially in regions of DNA accessible to micrococcal nuclease digestion. These regions are predominantly the linker regions between nucleosomes in chromatin. The alterations reported at polymerization and ligation steps of excision repair in the variant are therefore not associated with changes in the relative distributions of repair sites in linker and core particle regions of DNA.     

Clonal chromosome rearrangements in a fibroblast strain from a patient affected by xeroderma pigmentosum (complementation group C)

We report the results of DNA repair studies and cytogenetic investigations in a patient presenting acute phothosensitivity and cancerous skin lesions. In lymphocytes and fibroblasts a reduced level of unscheduled DNA synthesis after UV irradiation was found and the presence of xeroderma pigmentosum, complementation group C, mutation was demonstrated by complementation analysis. In lymphocyte and fibroblast cultures the frequency of spontaneous chromosome gaps and breaks was normal, whereas the frequency of chromosome rearrangements was higher than expected. In fibroblasts from the 4th to the 18th passage of the culture, 4 reciprocal translocations with a clonal distribution were identified. The rearranged chromosomes were Nos. 2, 13, 14 and 15, Nos. 2 and 13 being both involved in 3 different translocations with breakpoints at 2q21, 2q31, 2p23 and 13q31, 13q12 or 3. The biological significance of this finding is discussed in view of a possible correlation with the DNA repair defect and a possible relevance in tumor development of specific chromosome rearrangements.     

UV-induced base substitution mutations in a shuttle vector plasmid propagated in group C xeroderma pigmentosum cells.

To assess the contribution to mutagenesis of human DNA repair defects, the UV-irradiated shuttle vector plasmid pZ189 was propagated in fibroblasts derived from a xeroderma pigmentosum (XP) patient in DNA repair complementation group C. In comparison to results with DNA repair-proficient human cells (WI-38 VA13), UV-irradiated pZ189 propagated in the XP-C (XP4PA(SV)) cells showed fewer surviving plasmids and a higher frequency of mutated plasmids. Base sequence analysis of 67 mutated plasmids recovered from the XP-C cells revealed similar classes of point mutations and mutation spectrum, and a higher frequency of G:C to A:T transitions along with a lower frequency of transversions among plasmids with single or tandem mutations compared to plasmids recovered from the normal line. Most single-base substitution mutations (83%) occurred at G:C base pairs in which the 5'-adjacent base of the cytosine was thymine or cytosine. These results indicate that the DNA repair defects in XP-C, in comparison to data previously reported for XP-A, XP-D and XP-F, result in different UV survival and mutation frequency but in similar types of base substitution mutations.     

DNA polymerases eta and theta function in the same genetic pathway to generate mutations at A/T during somatic hypermutation of Ig genes

Somatic hypermutation of the Ig genes requires the activity of multiple DNA polymerases to ultimately introduce mutations at both A/T and C/G base pairs. Mice deficient for DNA polymerase eta (POLH) exhibited an approximately 80% reduction of the mutations at A/T, whereas absence of polymerase (POLQ) resulted in approximately 20% reduction of both A/T and C/G mutations. To investigate whether the residual A/T mutations observed in the absence of POLH are generated by POLQ and how these two polymerases might cooperate or compete with each other to generate A/T mutations, here we have established mice deficient for both POLH and POLQ. Polq(-/-)Polh(-/-) mice, however, did not show a further decrease of A/T mutations as compared with Polh(-/-) mice, suggesting that POLH and POLQ function in the same genetic pathway in the generation of these mutations. Frequent misincorporation of nucleotides, in particular opposite template T, is a known feature of POLH, but the efficiency of extension beyond the misincorporation differs significantly depending on the nature of the mispairing. Remarkably, we found that POLQ catalyzed extension more efficiently than POLH from all types of mispaired termini opposite A or T. Moreover, POLQ was able to extend mispaired termini generated by POLH albeit at a relatively low efficiency. These results reveal genetic and biochemical interactions between POLH and POLQ and suggest that POLQ might cooperate with POLH to generate some of the A/T mutations during the somatic hypermutation of Ig genes.     

SHMTool: a webserver for comparative analysis of somatic hypermutation datasets

The somatic hypermutation (SHM) of Immunoglobulin variable (V) regions is a key process in the generation of antibody diversity. The growing number of datasets of point mutations that occur during SHM in mice and humans often include comparisons between wild-type and individuals or strains genetically defective in the repair mechanisms that contribute to SHM. However, it has been difficult to compare the results of different studies because the analyses have not been standardized for criteria such as correction for base composition and the inclusion of unique mutations. If many mutations are involved, the analysis can also be time consuming. To overcome these problems and facilitate a standardized analysis and display of similar data, we present a webserver (SHMTool) for comparing SHM datasets, available at http://scb.aecom.yu.edu/shmtool.     

The cytopathicity of a simian immunodeficiency virus Mne variant is determined by mutations in Gag and Env.

Previous studies suggested that the rapidly replicating, highly cytopathic, syncytium-inducing (rapid-high/SI) phenotype of simian immunodeficiency virus Mne variants that evolved in macaques inoculated with a slowly replicating, minimally cytopathic, non-syncytium-inducing (slow-low/NSI) molecular clone was not solely the result of changes in the envelope surface protein (Env SU). To define the viral determinants responsible for the change in phenotype, we molecularly cloned a rapid-high/SI variant (designated SIVMne170) derived from the peripheral blood mononuclear cells (PBMCs) of a pig-tailed macaque that was inoculated with a slow-low/NSI molecular clone, SIVMneCL8. SIVMne170 was SI and replicated with faster kinetics and was more cytopathic than the parent SIVMneCL8 in CEMx174 cells. Additionally, SIVMne170 was more cytopathic for the CD4+ T-cell population than SIVMneCL8 in macaque PBMCs. An analysis of chimeric viruses constructed between the variant SIVMne170 and the parent virus SIVMneCL8 demonstrated that there are determinants encoded within both the 5' and 3' halves of SIVMne170 that independently contribute to its rapid-high/SI phenotype. As we previously observed with other SIVMne variants, the Env SU of SIVMne170 was important for syncytium induction but was not a key determinant of cytopathicity. By contrast, the intracellular domain of the envelope transmembrane protein (Env TM) contributed to both the SI and cytopathic properties of SIVMne170. We also found that the minimal determinant within the 5' half of SIVMne170 that conferred its rapid replication kinetics and cytopathicity mapped to the capsid- and nucleocapsid-encoding regions of gag. Together, these data demonstrate that mutations selected in Gag and Env TM intracytoplasmic tail influence the replication and cytopathicity of SIVMne variants that evolve in the host.     

Isolation by polymerase chain reaction of a cDNA whose product partially complements the ultraviolet sensitivity of xeroderma pigmentosum group C cells

A xeroderma pigmentosum (XP) cell line from complementation group C has been complemented to attain ultraviolet (UV) resistance and DNA repair proficiency, by transfection with a human expression cDNA library, followed by selection to UV resistance. We now show that the transfected cDNAs can be rescued from cellular DNA of a secondary transformant by its in vitro amplification using expression-vector-specific oligodeoxyribonucleotides as primers in a polymerase chain reaction. The amplified cDNAs were cloned into a mammalian expression vector. Their transfection into XP cells identified a single cDNA which specifically complemented the UV sensitivity of a group-C-derived cell line to the same partial UV-resistance levels exhibited by the transformant from which the cDNAs were rescued.     

Elevation of dCTP pools in xeroderma pigmentosum variant human fibroblasts alters the effects of DNA repair arrest by arabinofuranosyl cytosine

DNA excision repair inhibition by arabinofuranosyl cytosine (ara-C) or by ara-C/hydroxyurea (HU) was measured in log phase and confluent cultures of normal and xeroderma pigmentosium (XP)-variant human fibroblasts following insult by ultraviolet (UV) light (20 J/m²). Repair inhibition was determined by measuring the accumulation of DNA single-strand breaks/10⁸ daltons following cell culture exposure to ara-C or ara-C/HU in a series of 3 hr. pulses up ro 24 hr. after UV insult. Both normal and XP-variant derived cells showed a wide range of sensitivity to ara-C in log phase cells (0.2–9.4 breaks/10⁸ daltons DNA), although strand break accumulation was constant for each specific cell line. The same cells were more sensitive to ara-C/HU with a 2–14 fold increase in DNA strand breaks depending upon the cell line assayed. In confluent cultures of normal cells, maximum sensitivity to ara-C and ara-C/HU was achieved with similar levels of repair inhibition observed (16.1 and 16.5 breaks/10⁸ daltons, respectively). The same level of repair inhibition was observed in confulent XP-variants receiving ara-C/HU, but was reduced by 62–68% in cells treated with ara-C alone. Ara-C repair arrest was more rapidly reversed by competing concentrations of exogenous deoxycytidine (dCyd) in XP-variant compared to normal cells, especially in confluent cell cultures. In ara-C/HU treated cells, the level of dCyd reversal was reduced in the XP-variant when compared to cells exposed to ara-C alone. However, the same addition of HU had relatively little effect on dCyd reversal in normal cells. The measurements of dNTP levels indicate an elevated level of intracellular deoxycytosine triphosphate in XP-variant vs normal cells. The implications of these results are discussed as they relate to possible excision repair anomalies in the XP-variant.Abbreviations ara-C arabinofuranosul cytosine - dCTP deoxycytosine triphosphate - dCyd deoxycytidine - dNTP deoxynucleoside triphosphate - dT thymidine - HU hydroxyurea - XP xeroderma pigmentosium This research was sponsored jointly by the National Cancer Institute under Interagency Agreement #40-5-63, and the Office of Health and Environment Research, U. S. Department of Energy, under Contract W-7405-eng-26 with the Union Carbide Corporation.     

Sensitivity of excision repair in normal human,xeroderma pigmentosum variant and Cockayne's syndrome fibroblasts to inhibition by cytosine arabinoside

Inhibition of the gap-filling, polymerizing step of excision repair by 1-β-D-arabinofuranosylcytosine (ara-C) after irradiation with ultraviolet light in human diploid fibroblasts resulted in the formation of persistent DNA strand breaks in G₁, G₂, and plateau phase cells, but not in S phase cells. Addition of hydroxyurea to ara-C resulted in partial inhibition of repair in S phase cells. These observations can be explained either in terms of changing roles in repair for different DNA polymerases throughout the cell cycle or by the presence of a pool of deoxycytidine nucleotides during S phase equivalent to an external source of deoxycytidine at 50 μM concentration. A similar concentration dependence on ara-C was observed for inhibition of repair in normal human, xeroderma pigmentosum (XP) variant, and Cockayne's syndrome cells. Ara-C produced a similar number of breaks in normal and Cockayne's syndrome cells but slightly more in XP variant cells. Exonuclease III and S1 nuclease independently both degraded about 50% of the ₃H-thymidine incorporated into repaired regions in the presence of ara-C. Sequential digestion with both enzymes degraded nearly 90% of the repaired regions. These observations can be explained if excision repair proceeds by displacing the damaged strand so that both the ₃H-labeled patch and the damaged region are still ligated to high molecular weight DNA and compete for the same complementary strand during in vitro incubation with the nucleases. The amount of ₃H-thymidine incorporated in DNA by repair decreased with increasing concentrations of ara-C and hydroxyurea, suggesting that the incomplete patches became shorter under these conditions. Extrapolation of the digestion kinetics with exonuclease III permits an estimate of the normal patch size of about 100 nucleotides, consistent with previous estimates.