Biomedical subjects

T A Nagelhus

Publications and source records attributed to T A Nagelhus.

7 recordsLinked to original sources

PubMed1999

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.

Amino Acid Sequence↗

PubMed1998

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.

Alternative Splicing↗

PubMed1997

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.

Amino Acid Sequence↗

PubMed1997

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.

Alternative Splicing↗

PubMed1996

Fading correction for fluorescence quantitation in confocal microscopy.

Quantitative analysis in confocal microscopy meets with several problems such as fading of the fluorophore during scanning and attenuation of the fluorescence in thick tissue specimens. The present study reports a quantitative investigation of the enzyme uracil-DNA glycosylase (UDG), which removes uracils from DNA. For this study we developed a fading correction algorithm which takes into account both the number of prior scans in the specimen, and the differences in fading through the specimen from each prior scan, presumably due to differences in laser intensity at various axial distances from the focus position. On this point, our findings are in contrast with results reported in other well known papers, and indicate different fading at various distances from the laser focus position. The correction procedure can and should be established for the same specimen, but on a different part of the specimen from that used in the actual biological study. Calibration can thus be done on an unknown or inhomogenous object. For a series of confocal xy-scans through the immunostained cells, a corrected summation image representing total FITC-fluorescence related to UDG was obtained. Both noise removal and fading corrections were performed on each image in the series before the summation image was made. Estimates of total amounts of UDG localized in the cells and nuclei, respectively, could then be obtained. Measurement of the total cellular UDG-content by flow cytometry was also performed in order to make a comparison of the two methods for quantitative analysis. For both methods a range of approximately 4.5 was obtained between total UDG-content of cells at the 5 and 95 percentage points.

DNA Glycosylases↗

PubMed1995

Cell cycle regulation and subcellular localization of the major human uracil-DNA glycosylase.

The subcellular localization of the human DNA-repair enzyme uracil-DNA glycosylase from the UNG gene has been studied using flow cytometry and laser scanning confocal microscopy of freely cycling HeLa S3 cells. A two-parameter flow cytometric analysis using propidium iodide and UNG-specific antibodies demonstrated that total cellular UNG increased during the G1-phase and was approximately doubled in early S-phase compared to early G1. The UNG level was stable during the S-phase and increased further during G2, reaching a 2.8-fold level compared to early G1. This factor included differences in cell size and staining variabilities. These findings were confirmed using two-parameter confocal analysis of UNG/DNA and UNG/mitochondria at different stages of the cell cycle. Although the major fraction of UNG was associated with nuclei, we also observed distinctive staining associated with mitochondria and a more diffuse staining probably reflecting UNG in the cytosol. Furthermore, very little UNG staining was observed in nucleoli. The UNG level in different cell compartments varied at different stages of the cell cycle, and this variation was most pronounced in the nuclei. These results demonstrate that the gene product from the UNG gene is located within three subcellular compartments and that the distribution between these compartments varies during the cell cycle.

Cell Cycle↗

PubMed1993

Expression of the chondroitin sulphate proteoglycan molecular complex in six human melanoma xenograft lines studied by flow cytometry and immunohistochemistry.

The expression of the chondroitin sulphate proteoglycan (CSP) molecular complex in six human melanoma xenograft lines (BEX-t, COX-t, HUX-t, ROX-t, SAX-t, WIX-t) was studied by flow cytometry and immunohistochemistry using the monoclonal antibodies 9.2.27, ME31.3, G7A5, and NKI.M6. The two methods and the four antibodies gave consistent results. The six melanoma lines could be divided into three distinct groups of two lines each; expression was high in the HUX-t and ROX-t lines and intermediate in the BEX-t and SAX-t lines, whereas the COX-t and WIX-t lines were negative. The mean number of epitopes per cell for 9.2.27 was approximately twice as high as for ME31.3, G7A5, and NKI.M6 and was estimated to range from 0.8 +/- 0.1 x 10(5) to 1.9 +/- 0.2 x 10(5) in the positive xenograft lines. The expression of the CSP complex was heterogeneous. The immunofluorescence histograms measured by flow cytometry were therefore broad for all tumour lines. A significant fraction of the HUX-t cells was negative or weakly stained. These cells appeared as clear negative patches in the immunohistochemical preparations. Moreover, most morphologically intact tumour cells adjacent to necrotic areas did not show significant expression of the CSP complex, irrespective of tumour line. These cells were probably hypoxic and thus resistant to radiation therapy. The expression of the CSP complex in the xenograft lines was similar to that reported for melanoma in man.

Animals↗