Mre11 exonuclease activity removes the chain-terminating nucleoside analog gemcitabine from the nascent strand during DNA replication.

The Mre11 nuclease is involved in early responses to DNA damage, often mediated by its role in DNA end processing. MRE11 mutations and aberrant expression are associated with carcinogenesis and cancer treatment outcomes. While, in recent years, progress has been made in understanding the role of Mre11 nuclease activities in DNA double-strand break repair, their role during replication has remained elusive. The nucleoside analog gemcitabine, widely used in cancer therapy, acts as a replication chain terminator; for a cell to survive treatment, gemcitabine needs to be removed from replicating DNA. Activities responsible for this removal have, so far, not been identified. We show that Mre11 3' to 5' exonuclease activity removes gemcitabine from nascent DNA during replication. This contributes to replication progression and gemcitabine resistance. We thus uncovered a replication-supporting role for Mre11 exonuclease activity, which is distinct from its previously reported detrimental role in uncontrolled resection in recombination-deficient cells.

Different organization of base excision repair of uracil in DNA in nuclei and mitochondria and selective upregulation of mitochondrial uracil-DNA glycosylase after oxidative stress.

Oxidative stress in the brain may cause neuro-degeneration, possibly due to DNA damage. Oxidative base lesions in DNA are mainly repaired by base excision repair (BER). The DNA glycosylases Nei-like DNA glycosylase 1 (NEIL1), Nei-like DNA glycosylase 2 (NEIL2), mitochondrial uracil-DNA glycosylase 1 (UNG1), nuclear uracil-DNA glycosylase 2 (UNG2) and endonuclease III-like 1 protein (NTH1) collectively remove most oxidized pyrimidines, while 8-oxoguanine-DNA glycosylase 1 (OGG1) removes oxidized purines. Although uracil is the main substrate of uracil-DNA glycosylases UNG1 and UNG2, these proteins also remove the oxidized cytosine derivatives isodialuric acid, alloxan and 5-hydroxyuracil. UNG1 and UNG2 have identical catalytic domain, but different N-terminal regions required for subcellular sorting. We demonstrate that mRNA for UNG1, but not UNG2, is increased after hydrogen peroxide, indicating regulatory effects of oxidative stress on mitochondrial BER. To examine the overall organization of uracil-BER in nuclei and mitochondria, we constructed cell lines expressing EYFP (enhanced yellow fluorescent protein) fused to UNG1 or UNG2. These were used to investigate the possible presence of multi-protein BER complexes in nuclei and mitochondria. Extracts from nuclei and mitochondria were both proficient in complete uracil-BER in vitro. BER assays with immunoprecipitates demonstrated that UNG2-EYFP, but not UNG1-EYFP, formed complexes that carried out complete BER. Although apurinic/apyrimidinic site endonuclease 1 (APE1) is highly enriched in nuclei relative to mitochondria, it was apparently the major AP-endonuclease required for BER in both organelles. APE2 is enriched in mitochondria, but its possible role in BER remains uncertain. These results demonstrate that nuclear and mitochondrial BER processes are differently organized. Furthermore, the upregulation of mRNA for mitochondrial UNG1 after oxidative stress indicates that it may have an important role in repair of oxidized pyrimidines.

The 118 A > G polymorphism in the human mu-opioid receptor gene may increase morphine requirements in patients with pain caused by malignant disease.

BACKGROUND: Dispositions for genes encoding opioid receptors may explain some variability in morphine efficacy. Experimental studies show that morphine and morphine-6-glucuronide are less effective in individuals carrying variant alleles caused by the 118 A > G polymorphism in the mu-opioid receptor gene (OPRM1). The purpose of the study was to investigate whether this and other genetic polymorphisms in OPRM1 influence the efficacy of morphine in cancer pain patients. METHODS: We screened 207 cancer pain patients on oral morphine treatment for four frequent OPRM1 gene polymorphisms. The polymorphisms were the -172 G > T polymorphism in the 5'untranslated region of exon 1, the 118 A > G polymorphism in exon 1, and the IVS2 + 31 G > A and IVS2 + 691 G > C polymorphisms, both in intron 2. Ninety-nine patients with adequately controlled pain were included in an analysis comparing morphine doses and serum concentrations of morphine and morphine metabolites in the different genotypes for the OPRM1 polymorphisms. RESULTS: No differences related to the -172 G > T, the IVS2 + 31 G > A and the IVS2 + 691 G > C polymorphisms were observed. Patients homozygous for the variant G allele of the 118 A > G polymorphism (n = 4) needed more morphine to achieve pain control, compared to heterozygous (n = 17) and homozygous wild-type (n = 78) individuals. This difference was not explained by other factors such as duration of morphine treatment, performance status, time since diagnosis, time until death, or adverse symptoms. CONCLUSION: Patients homozygous for the 118 G allele of the mu-opioid receptor need higher morphine doses to achieve pain control. Thus, genetic variation at the gene encoding the mu-opioid receptor contributes to variability in patients' responses to morphine.

A gel electrophoresis method for detection of mitochondrial DNA mutation (3243 tRNA(Leu (UUR))) applied to a Norwegian family with diabetes mellitus and hearing loss.

Blood cells of selected patients from a large Norwegian family with maternally transmitted diabetes mellitus, hearing loss and muscular dysfunction were screened for possible A3243G mutation tRNA(Leu (UUR)) in mitochondrial DNA. We selected 7 patients from 3 of the 4 generations of the family and 10 unrelated healthy control subjects for mutation analysis using denaturing gradient gel electrophoresis (DGGE) and both manual and automated DNA sequencing. The A3243G mutation was found in peripheral blood cells of all 7 patients, but in none of the controls. The mutation was in the form of heteroplasmy and the amount of mutant DNA was found to be between 10% and 35% of total mtDNA in individual patients. This is the first report of a Norwegian family with maternally inherited diabetes and hearing loss carrying the A3243G mutation in mitochondrial DNA.

Sequence variations in the UDP-glucuronosyltransferase 2B7 (UGT2B7) gene: identification of 10 novel single nucleotide polymorphisms (SNPs) and analysis of their relevance to morphine glucuronidation in cancer patients.

We have screened a cohort of 239 Norwegian cancer patients for sequence variation in the coding and regulatory regions of the UDP-glucuronosyltransferase 2B7 gene (UGT2B7) and analyzed the impact of gene variants on morphine glucuronidation in vivo. In all, 12 single nucleotide polymorphisms (SNPs) were identified, 10 of which have not been previously described. Only one SNP causes a change in amino acid sequence (H268Y). Seven UGT2B7 genotypes were observed and three main haplotypes predicted. There was no correlation between UGT2B7 genotype or haplotype and morphine glucuronide to morphine serum ratios among 175 patients who received chronic oral morphine therapy, and who had normal renal and hepatic function. The apparent lack of functional polymorphisms fits well with the near unimodal, but broad, distributions of the ratios (morphine 3-glucuronide/morphine: 6.4-309.2; morphine 6-glucuronide/morphine: 0.5-72.8). Our results suggest that factors other than UGT2B7 polymorphism may be more deciding for the variability in morphine glucuronide to morphine serum ratios.

Properties and functions of human uracil-DNA glycosylase from the UNG gene.

The human UNG-gene at position 12q24.1 encodes nuclear (UNG2) and mitochondrial (UNG1) forms of uracil-DNA glycosylase using differentially regulated promoters, PA and PB, and alternative splicing to produce two proteins with unique N-terminal sorting sequences. PCNA and RPA co-localize with UNG2 in replication foci and interact with N-terminal sequences in UNG2. Mitochondrial UNG1 is processed to shorter forms by mitochondrial processing peptidase (MPP) and an unidentified mitochondrial protease. The common core catalytic domain in UNG1 and UNG2 contains a conserved DNA binding groove and a tight-fitting uracil-binding pocket that binds uracil only when the uracil-containing nucleotide is flipped out. Certain single amino acid substitutions in the active site of the enzyme generate DNA glycosylases that remove either thymine or cytosine. These enzymes induce cytotoxic and mutagenic abasic (AP) sites in the E. coli chromosome and were used to examine biological consequences of AP sites. It has been assumed that a major role of the UNG gene product(s) is to repair mutagenic U:G mispairs caused by cytosine deamination. However, one major role of UNG2 is to remove misincorporated dUMP residues. Thus, knockout mice deficient in Ung activity (Ung-/- mice) have only small increases in GC-->AT transition mutations, but Ung-/- cells are deficient in removal of misincorporated dUMP and accumulate approximately 2000 uracil residues per cell. We propose that BER is important both in the prevention of cancer and for preserving the integrity of germ cell DNA during evolution.

Cell-specific enhancement of doxorubicin toxicity in human tumour cells by docosahexaenoic acid.

We examined the cytotoxicity of doxorubicin alone, or in combination with docosahexaenoic acid (22:6 n-3), in glioblastoma cell lines A-172 and U-87 MG and bronchial carcinoma cell lines A-427 and SK-LU-1. For both glioblastoma cell lines we found an enhanced cytotoxicity of doxorubicin when given with concentrations of docosahexaenoic acid that alone are non-toxic. In SK-LU-1 cells no such enhancement was observed, whereas a small increase was observed for A-427 cells. The enhanced cytotoxicity in glioblastoma cells was not caused by lipid peroxidation products. In A-427 cells, however, the modest potentiation could be explained by the formation of cytotoxic lipid peroxidation products. Se-glutathione peroxidase activity increased after doxorubicin exposure and even more after addition of Na-selenite, but this did not reduce the cytotoxicity of doxorubicin. These results demonstrated that the mechanisms of enhancement of cytotoxicity by docosahexaenoic acid are complex and cell-specific and do not require increased lipid peroxidation.

Analysis of uracil DNA glycosylase in human colorectal cancer.

Uracil DNA glycosylase (UDG) is responsible for the removal of uracil present in DNA after cytosine deamination or misincorporation during replication. Colorectal cancer is widely treated with 5-FU, which leads to thymidylate synthase inhibition; this accounts for increased dUTP intracellular pools and subsequent uracil incorporation into DNA. Uracil misincorporation has also been implicated in the link between folate deficiency and colorectal cancer risk. As there is no information on UDG in colorectal cancer, this study characterized UDG activity and protein expression in a panel of 20 colorectal tumors and 6 colorectal cell lines. UDG activity in colorectal tissue is widely variable and it is statistically higher in tumor tissue (P=0.013) compared to normal bowel. Tumor versus normal activity ratios ranged from 0.49 to 2.2 (median 1.13). Among the six colorectal cell lines tested, UDG activity varied from 40 to 68 units and was markedly (1.7-fold) higher than in tumor tissue (P<0.0001). In both colorectal tissues and cell lines, UDG was expressed as both 29 kDa and 35 kDa forms. Total protein expression varied 3.2-fold in cell lines; variability was also found between patients and between normal and tumoral tissue for the same patient. This study demonstrates UDG protein and functional activity in human colorectal tumors and cell lines. The high tumor:normal tissue ratio supports further interest in base excision repair, through UDG, as a potential source of fluoropyrimidine resistance in colorectal cancer.

Sequence variation in the human uracil-DNA glycosylase (UNG) gene.

Spontaneous deamination of cytosine results in a premutagenic G:U mismatch that may result in a GC-->AT transition during replication. The human UNG-gene encodes the major uracil-DNA glycosylase (UDG or UNG) which releases uracil from DNA, thus, initiating base excision repair to restore the correct DNA sequence. Bacterial and yeast mutants lacking the homologous UDG exhibit elevated spontaneous mutation frequencies. Hence, mutations in the human UNG gene could presumably result in a mutator phenotype. We screened all seven exons including exon-intron boundaries, both promoters, and one intron of the UNG gene and identified considerable sequence variation in cell lines derived from normal fibroblasts and tumour tissue. None of the sequence variants was accompanied by significantly reduced UDG activity. In the UNG gene from 62 sources, we identified 12 different variant alleles, with allele frequencies ranging from 0.01 to 0.23. We identified one variant allele per 3.8kb in non-coding regions, but none in the coding region of the gene. In promoter B we identified four different variants. A substitution within an AP2 element was observed in tumour cell lines only and had an allele frequency of 0.10. Introduction of this substitution into chimaeric promoter-luciferase constructs affected transcription from the promoter. UDG-activity varied little in fibroblasts, but widely between tumour cell lines. This variation did not however correlate with the presence of any of the variant alleles. In conclusion, mutations affecting the function of human UNG gene are seemingly infrequent in human tumour cell lines.

Repair of chromosomal abasic sites in vivo involves at least three different repair pathways.

We introduced multiple abasic sites (AP sites) in the chromosome of repair-deficient mutants of Escherichia coli, in vivo, by expressing engineered variants of uracil-DNA glycosylase that remove either thymine or cytosine. After introduction of AP sites, deficiencies in base excision repair (BER) or recombination were associated with strongly enhanced cytotoxicity and elevated mutation frequencies, selected as base substitutions giving rifampicin resistance. In these strains, increased fractions of transversions and untargeted mutations were observed. In a recA mutant, deficient in both recombination and translesion DNA synthesis (TLS), multiple AP sites resulted in rapid cell death. Preferential incorporation of dAMP opposite a chromosomal AP site ('A rule') required UmuC. Furthermore, we observed an 'A rule-like' pattern of spontaneous mutations that was also UmuC dependent. The mutation patterns indicate that UmuC is involved in untargeted mutations as well. In a UmuC-deficient background, a preference for dGMP was observed. Spontaneous mutation spectra were generally strongly dependent upon the repair background. In conclusion, BER, recombination and TLS all contribute to the handling of chromosomal AP sites in E.coli in vivo.

Base excision repair of DNA in mammalian cells.

Base excision repair (BER) of DNA corrects a number of spontaneous and environmentally induced genotoxic or miscoding base lesions in a process initiated by DNA glycosylases. An AP endonuclease cleaves at the 5' side of the abasic site and the repair process is subsequently completed via either short patch repair or long patch repair, which largely require different proteins. As one example, the UNG gene encodes both nuclear (UNG2) and mitochondrial (UNG1) uracil DNA glycosylase and prevents accumulation of uracil in the genome. BER is likely to have a major role in preserving the integrity of DNA during evolution and may prevent cancer.

Uracil-DNA glycosylase (UNG)-deficient mice reveal a primary role of the enzyme during DNA replication.

Gene-targeted knockout mice have been generated lacking the major uracil-DNA glycosylase, UNG. In contrast to ung- mutants of bacteria and yeast, such mice do not exhibit a greatly increased spontaneous mutation frequency. However, there is only slow removal of uracil from misincorporated dUMP in isolated ung-/- nuclei and an elevated steady-state level of uracil in DNA in dividing ung-/- cells. A backup uracil-excising activity in tissue extracts from ung null mice, with properties indistinguishable from the mammalian SMUG1 DNA glycosylase, may account for the repair of premutagenic U:G mispairs resulting from cytosine deamination in vivo. The nuclear UNG protein has apparently evolved a specialized role in mammalian cells counteracting U:A base pairs formed by use of dUTP during DNA synthesis.

Analysis of uracil-DNA glycosylases from the murine Ung gene reveals differential expression in tissues and in embryonic development and a subcellular sorting pattern that differs from the human homologues.

The murine UNG: gene encodes both mitochondrial (Ung1) and nuclear (Ung2) forms of uracil-DNA glyco-sylase. The gene contains seven exons organised like the human counterpart. While the putative Ung1 promoter (P(B)) and the human P(B) contain essentially the same, although differently organised, transcription factor binding elements, the Ung2 promoter (P(A)) shows limited homology to the human counterpart. Transient transfection of chimaeric promoter-luciferase constructs demonstrated that both promoters are functional and that P(B) drives transcription more efficiently than P(A). mRNAs for Ung1 and Ung2 are found in all adult tissues analysed, but they are differentially expressed. Furthermore, transcription of both mRNA forms, particularly Ung2, is induced in mid-gestation embryos. Except for a strong conservation of the 26 N-terminal residues in Ung2, the subcellular targeting sequences in the encoded proteins have limited homology. Ung2 is transported exclusively to the nucleus in NIH 3T3 cells as expected. In contrast, Ung1 was sorted both to nuclei and mitochondria. These results demonstrate that although the catalytic domain of uracil-DNA glycosylase is highly conserved in mouse and man, regulatory elements in the gene and subcellular sorting sequences in the proteins differ both structurally and functionally, resulting in altered contribution of the isoforms to total uracil-DNA glycosylase activity.

Uracil-DNA glycosylase-DNA substrate and product structures: conformational strain promotes catalytic efficiency by coupled stereoelectronic effects.

Enzymatic transformations of macromolecular substrates such as DNA repair enzyme/DNA transformations are commonly interpreted primarily by active-site functional-group chemistry that ignores their extensive interfaces. Yet human uracil-DNA glycosylase (UDG), an archetypical enzyme that initiates DNA base-excision repair, efficiently excises the damaged base uracil resulting from cytosine deamination even when active-site functional groups are deleted by mutagenesis. The 1.8-A resolution substrate analogue and 2.0-A resolution cleaved product cocrystal structures of UDG bound to double-stranded DNA suggest enzyme-DNA substrate-binding energy from the macromolecular interface is funneled into catalytic power at the active site. The architecturally stabilized closing of UDG enforces distortions of the uracil and deoxyribose in the flipped-out nucleotide substrate that are relieved by glycosylic bond cleavage in the product complex. This experimentally defined substrate stereochemistry implies the enzyme alters the orientation of three orthogonal electron orbitals to favor electron transpositions for glycosylic bond cleavage. By revealing the coupling of this anomeric effect to a delocalization of the glycosylic bond electrons into the uracil aromatic system, this structurally implicated mechanism resolves apparent paradoxes concerning the transpositions of electrons among orthogonal orbitals and the retention of catalytic efficiency despite mutational removal of active-site functional groups. These UDG/DNA structures and their implied dissociative excision chemistry suggest biology favors a chemistry for base-excision repair initiation that optimizes pathway coordination by product binding to avoid the release of cytotoxic and mutagenic intermediates. Similar excision chemistry may apply to other biological reaction pathways requiring the coordination of complex multistep chemical transformations.

Post-replicative base excision repair in replication foci.

Base excision repair (BER) is initiated by a DNA glycosylase and is completed by alternative routes, one of which requires proliferating cell nuclear antigen (PCNA) and other proteins also involved in DNA replication. We report that the major nuclear uracil-DNA glycosylase (UNG2) increases in S phase, during which it co-localizes with incorporated BrdUrd in replication foci. Uracil is rapidly removed from replicatively incorporated dUMP residues in isolated nuclei. Neutralizing antibodies to UNG2 inhibit this removal, indicating that UNG2 is the major uracil-DNA glycosylase responsible. PCNA and replication protein A (RPA) co-localize with UNG2 in replication foci, and a direct molecular interaction of UNG2 with PCNA (one binding site) and RPA (two binding sites) was demonstrated using two-hybrid assays, a peptide SPOT assay and enzyme-linked immunosorbent assays. These results demonstrate rapid post-replicative removal of incorporated uracil by UNG2 and indicate the formation of a BER complex that contains UNG2, RPA and PCNA close to the replication fork.

Acrolein cytotoxicity and glutathione depletion in n-3 fatty acid sensitive- and resistant human tumor cells.

Acrolein is a highly reactive unsaturated aldehyde formed endogenously and present in the environment. Acrolein efficiently reduces glutathione-contents and is highly cytotoxic in two lung carcinoma cell lines (A-427 and SK-LU-1) and the glioblastoma cell line A-172. A-427, which has the lowest GSH content of the cell lines, is also more sensitive to growth inhibition and more depleted in GSH after acrolein exposure. A-427 is also highly sensitive to docosahexaenoic acid (22:6 n-3, DHA) and acrolein potentiates the cytotoxic effect of DHA in this cell line, but not in the DHA-resistant cell lines SK-LU-1 and A-172. Surprisingly, the cytotoxic effect of acrolein was partially reversed by vitamin E, selenite and 2-phenyl-1,2-benzisoselenazol-3(2H)-one (ebselen, a Se-glutathione peroxidase mimic) in A-427 cells, but not in SK-LU-1 and A-172 cells. Using the TUNEL assay a strong nuclear fluorescence was observed in DHA-treated A-427 cells, indicating death by apoptosis, whereas acrolein apparently did not induce apoptosis.

Nuclear and mitochondrial splice forms of human uracil-DNA glycosylase contain a complex nuclear localisation signal and a strong classical mitochondrial localisation signal, respectively.

Nuclear (UNG2) and mitochondrial (UNG1) forms of human uracil-DNA glycosylase are both encoded by the UNG gene but have different N-terminal sequences. We have expressed fusion constructs of truncated or site-mutated UNG cDNAs and green fluorescent protein cDNA and studied subcellular sorting. The unique 44 N-terminal amino acids in UNG2 are required, but not sufficient, for complete sorting to nuclei. In this part the motif R17K18R19is essential for sorting. The complete nuclear localization signal (NLS) in addition requires residues common to UNG2 and UNG1 within the 151 N-terminal residues. Replacement of certain basic residues within this region changed the pattern of subnuclear distribution of UNG2. The 35 unique N-terminal residues in UNG1 constitute a strong and complete mitochondrial localization signal (MLS) which when placed at the N-terminus of UNG2 overrides the NLS. Residues 11-28 in UNG1 have the potential of forming an amphiphilic helix typical of MLSs and residues 1-28 are essential and sufficient for mitochondrial import. These results demonstrate that UNG1 contains a classical and very strong MLS, whereas UNG2 contains an unusually long and complex NLS, as well as subnuclear targeting signals in the region common to UNG2 and UNG1.

Human mitochondrial uracil-DNA glycosylase preform (UNG1) is processed to two forms one of which is resistant to inhibition by AP sites.

The preform of human mitochondrial uracil-DNA glycosylase (UNG1) contains 35 N-terminal residues required for mitochondrial targeting. We have examined processing of human UNG1 expressed in insect cells and processing in vitro by human mitochondrial extracts . In insect cells we detected a major processed form lacking 29 of the 35 unique N-terminal residues (UNG1Delta29, 31 kDa) and two minor forms lacking the 75 and 77 N-terminal residues, respectively (UNG1Delta75 and UNG1Delta77, 26 kDa). Purified UNG1Delta29 was effectively cleaved in vitro to a fully active 26 kDa form by human mitochondrial extracts. Furthermore, endogenous forms of 31 and 26 kDa were also observed in HeLa mitochondrial extracts. The sequences at the cleavage sites, as identified by peptide sequencing, were compatible with the known specificity of mitochondrial processing peptidase (MPP). However, in vitro cleavage of UNG1Delta29 by mitochondrial extracts did not require divalent cations and was stimulated by EDTA, indicating the involvement of a processing peptidase distinct from MPP at the second site. Interestingly, while UNG1Delta29 generally has the typical properties reported for other uracil-DNA glycosylases, it is not inhibited by apurinic/apyrimidinic sites. Our results indicate that the preform of human mitochondrial uracil-DNA glycosylase is processed to distinctly different forms lacking 29 or 75/77 N-terminal residues, respectively.

Base excision repair initiation revealed by crystal structures and binding kinetics of human uracil-DNA glycosylase with DNA.

Three high-resolution crystal structures of DNA complexes with wild-type and mutant human uracil-DNA glycosylase (UDG), coupled kinetic characterizations and comparisons with the refined unbound UDG structure help resolve fundamental issues in the initiation of DNA base excision repair (BER): damage detection, nucleotide flipping versus extrahelical nucleotide capture, avoidance of apurinic/apyrimidinic (AP) site toxicity and coupling of damage-specific and damage-general BER steps. Structural and kinetic results suggest that UDG binds, kinks and compresses the DNA backbone with a 'Ser-Pro pinch' and scans the minor groove for damage. Concerted shifts in UDG simultaneously form the catalytically competent active site and induce further compression and kinking of the double-stranded DNA backbone only at uracil and AP sites, where these nucleotides can flip at the phosphate-sugar junction into a complementary specificity pocket. Unexpectedly, UDG binds to AP sites more tightly and more rapidly than to uracil-containing DNA, and thus may protect cells sterically from AP site toxicity. Furthermore, AP-endonuclease, which catalyzes the first damage-general step of BER, enhances UDG activity, most likely by inducing UDG release via shared minor groove contacts and flipped AP site binding. Thus, AP site binding may couple damage-specific and damage-general steps of BER without requiring direct protein-protein interactions.