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.

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.

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.

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.

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.

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.

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.

A sequence in the N-terminal region of human uracil-DNA glycosylase with homology to XPA interacts with the C-terminal part of the 34-kDa subunit of replication protein A.

Uracil-DNA glycosylase releases free uracil from DNA and initiates base excision repair for removal of this potentially mutagenic DNA lesion. Using the yeast two-hybrid system, human uracil-DNA glycosylase encoded by the UNG gene (UNG) was found to interact with the C-terminal part of the 34-kDa subunit of replication protein A (RPA2). No interaction with RPA4 (a homolog of RPA2), RPA1, or RPA3 was observed. A sandwich enzyme-linked immunosorbent assay with trimeric RPA and the two-hybrid system both demonstrated that the interaction depends on a region in UNG localized between amino acids 28 and 79 in the open reading frame. In this part of UNG a 23-amino acid sequence has a significant homology to the RPA2-binding region of XPA, a protein involved in damage recognition in nucleotide excision repair. Trimeric RPA did not enhance the activity of UNG in vitro on single- or double-stranded DNA. A part of the N-terminal region of UNG corresponding in size to the complete presequence was efficiently removed by proteinase K, leaving the proteinase K-resistant compact catalytic domain intact and fully active. These results indicate that the N-terminal part constitutes a separate structural domain required for RPA binding and suggest a possible function for RPA in base excision repair.

Nuclear and mitochondrial uracil-DNA glycosylases are generated by alternative splicing and transcription from different positions in the UNG gene.

A distinct nuclear form of human uracil-DNA glycosylase [UNG2, open reading frame (ORF) 313 amino acid residues] from the UNG gene has been identified. UNG2 differs from the previously known form (UNG1, ORF 304 amino acid residues) in the 44 amino acids of the N-terminal sequence, which is not necessary for catalytic activity. The rest of the sequence and the catalytic domain, altogether 269 amino acids, are identical. The alternative N-terminal sequence in UNG2 arises by splicing of a previously unrecognized exon (exon 1A) into a consensus splice site after codon 35 in exon 1B (previously designated exon 1). The UNG1 sequence starts at codon 1 in exon 1B and thus has 35 amino acids not present in UNG2. Coupled transcription/translation in rabbit reticulocyte lysates demonstrated that both proteins are catalytically active. Similar forms of UNG1 and UNG2 are expressed in mouse which has an identical organization of the homologous gene. Constructs that express fusion products of UNG1 or UNG2 and green fluorescent protein (EGFP) were used to study the significance of the N-terminal sequences in UNG1 and UNG2 for subcellular targeting. After transient transfection of HeLa cells, the pUNG1-EGFP-N1 product colocalizes with mitochondria, whereas the pUNG2-EGFP-N1 product is targeted exclusively to nuclei.

Effects of anti-CD18 and LPS on CD14 expression on human monocytes.

In this study we show that the cytokine stimulatory effect of LPS on human monocytes is enhanced by addition of monoclonal antibodies against CD18 (alpha CD18 MoAbs). Incubation of monocytes with alpha CD18 MoAbs overnight increased the CD14 expression as detected by Leu-M3, but not with My-4. These results indicate that CD18 participates in LPS-induced TNF-alpha production as well as in regulating CD14 expression on monocytes. Addition of LPS to monocytes resulted in a reduction in the CD14 expression after 1/2, 1, 2 and 4 h, but increased CD14 expression was seen after LPS stimulation overnight. By doing double labelling of the monocyte population for CD14 and CD16 it was found that the reduction in CD14 expression occurred in the CD14+/CD16+ sub-population, while the increase in CD14 expression was seen in both the CD14+/CD16- and the CD14+/CD16+ cells. alpha CD14 MoAbs that were able to inhibit LPS-induced cytokine production from monocytes (3C10 and My-4) were considerably less able to detect the increase in CD14 expression after LPS stimulation than alpha CD14 MoAbs that did not inhibit LPS-induced cytokine production (Leu-M3 and alpha CD14Serva). Our data indicate that My-4 and Leu-M3 define two populations of CD14+ cells on LPS stimulated human monocytes.

Soluble CD14 from urine copurifies with a potent inducer of cytokines.

Here we report that soluble CD14 isolated from the urine of nephrotic patients (uCD14) contains a potent cytokine inducing activity. CD14 derived from urine appeared to consist of two major polypeptides of about 54 and 48 kDa. In uCD14 isolated from three different nephrotic patients the cytokine-inducing activity appeared to co-migrate with the 48-kDa polypeptide which upon sequencing had the same N-terminal sequence as native CD14. Treatment of human monocytes and the human astrocytoma cell line U373 with uCD14 resulted in a strong secretion of tumor necrosis factor (TNF) and interleukin-6, respectively. The cytokine-inducing activity of the uCD14 preparations was unaffected by the absence of serum. This is in contrast to the activation of human monocytes and U373 cells by lipopolysaccharide (LPS) which is highly dependent on the presence of serum. The cytokine-inducing activity was not affected by LPS-binding protein (LBP) or polyclonal rabbit antibodies against LBP. The TNF-inducing activity of uCD14 was also heat labile in contrast to the cytokine-inducing activity of LPS, which was relatively heat resistant. The results suggest that CD14 may exist in at least two forms of which one is involved in cytokine induction.

Characterization of binding and TNF-alpha-inducing ability of chitosans on monocytes: the involvement of CD14.

Chitosans with different chemical composition were found to induce TNF-alpha production from human monocytes. Their ability to induce TNF-alpha was found to be highly dependent on neutral-solubility and molecular weight. Monoclonal antibodies against CD14 inhibited TNF-alpha production from monocytes stimulated with neutral-soluble chitosans. Binding studies indicated that lipopolysaccharides (LPS) and neutral-soluble chitosans share a binding site on monocytes which involves CD14. TNF-alpha production from monocytes stimulated with chitosans was dependent on serum. LPS-binding protein (LBP) enhanced the chitosan-induced TNF-alpha production only to a minor degree, suggesting that serum proteins other than LBP play an important role in the stimulatory effect.

Similar mechanisms of action of defined polysaccharides and lipopolysaccharides: characterization of binding and tumor necrosis factor alpha induction.

Little has been reported about the effects of different polysaccharides on cytokine production from human monocytes. In this study, we show that several well-defined polysaccharides, including polymers with different sizes of beta 1-4-linked D-mannuronic acid (poly-M, high-M alginate, and M-blocks) and cellulose oxidized in the C-6 position, induced human monocytes to produce tumor necrosis factor alpha (TNF-alpha). Poly-M was the most efficient polysaccharide tested and, on a weight basis, was approximately as efficient as lipopolysaccharide (LPS) from Escherichia coli. TNF-alpha production was shown to depend strongly on the molecular weights of poly-M and high-M alginate, with maximal TNF-alpha production occurring at molecular weights above 50,000 and 200,000, respectively. G-blocks, alpha 1-4-linked L-guluronic acid polymers that did not induce cytokine production from monocytes, reduced the cytokine production induced by the beta 1-4-linked polyuronic acids and LPS. Furthermore, both G-blocks and LPS were found to inhibit the binding of poly-M to monocytes, as measured by flow cytometry. In addition, we found that the binding of LPS to monocytes was inhibited by G-blocks, M-blocks, and poly-M. Our results indicate that beta 1-4-linked polyuronic acids and LPS may stimulate monocytes to produce TNF-alpha by similar mechanisms and may bind to a common receptor.

The involvement of CD14 in stimulation of cytokine production by uronic acid polymers.

In this study the molecular mechanisms behind the stimulatory activities of the uronic acid polymers poly mannuronic acid (poly M), high M alginate and oxidized cellulose (C60XY) were investigated and compared with lipopolysaccharide (LPS). The cytokine-inducing abilities of the uronic acid polymers and LPS were examined on CD14-positive human monocytes and CD14-negative U373 astrocytoma cells. It was found that LPS induced monocytes and U373 cells to produce tumor necrosis factor (TNF) and interleukin(IL)-6, respectively, by different mechanisms. The poly uronic acids induced monocytes to produce TNF, but with 100-1000 times less potency compared to LPS. On U373 cells, LPS at concentrations > or = 32 ng/ml resulted in a dose-related IL-6 production, whereas the poly uronic acids had negligible effects even at 1 mg/ml. The binding data demonstrate that only the CD14-positive monocytes in the peripheral blood mononuclear cells population bound poly M. Furthermore, poly M was found to bind to CD14 in the presence of serum. Antibodies against CD14 also inhibited the TNF-inducing activity of the three uronic acid polymers tested. In conclusion, these results demonstrate that uronic acid polymers induce TNF production through mechanisms which involve CD14.

Induction of cytokine production from human monocytes stimulated with alginate.

Alginates are polysaccharides with gel-forming properties composed of 1,4-linked beta-D-mannuronic acid (M), alpha-L-guluronic acid (G), and alternating (MG) blocks. Alginate can be used as a matrix for implanted cells in vivo. In this study, we have examined the ability of alginates and their components to stimulate human monocytes to produce tumor necrosis factor-alpha, interleukin-6, and interleukin-1. Alginates stimulated the monocytes to produce high levels of all three cytokines. Low G alginates were approximately 10 times more potent in inducing cytokine production compared with high G alginates. The M-blocks and the MG-blocks, but not the G-blocks, stimulated the cytokine production. The results demonstrate that the mannuronic acid residues are the active cytokine inducers in alginates.