Activity of uracil‐DNA glycosylase in different rat tissues and in regenerating rat liver

Schwartz

Journal of Biological Chemistry

et al. 2004

A chronic imbalance in DNA precursors, caused by one-carbon metabolism impairment, can result in a deficiency of DNA repair and increased DNA damage. Although indirect evidence suggests that DNA damage plays a role in neuronal apoptosis and in the pathogenesis of neurodegenerative disorders, the underlying mechanisms are poorly understood. In particular, very little is known about the role of base excision repair of misincorporated uracil in neuronal survival. To test the hypothesis that repair of DNA damage associated with uracil misincorporation is critical for neuronal survival, we employed an antisense (AS) oligonucleotide directed against uracil-DNA glycosylase encoded by the UNG gene to deplete UNG in cultured rat hippocampal neurons. AS, but not a scrambled control oligonucleotide, induced apoptosis, which was associated with DNA damage analyzed by comet assay and up-regulation of p53. UNG mRNA and protein levels were decreased within 30 min and were undetectable within 6 -9 h of exposure to the UNG AS oligonucleotide. Whereas UNG expression is significantly higher in proliferating as compared with nonproliferating cells, such as neurons, the levels of UNG mRNA were increased in brains of cystathionine ␤-synthase knockout mice, a model for hyperhomocysteinemia, suggesting that one-carbon metabolism impairment and uracil misincorporation can induce the up-regulation of UNG expression.

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 82%

See 1 more Smart Citation

Suppression of Uracil-DNA Glycosylase Induces Neuronal Apoptosis

Schwartz

Journal of Biological Chemistry

et al. 2004

“…Although all human cells and tissues investigated have measurable UNG activities, significant interindividual as well as interorgan variation in activities has been observed in extracts from human adult tissues [21,22]. Furthermore, stimulation of peripheral lymphocytes with phytohemagglutinin, and studies of cells with different proliferation rates have demonstrated a correlation between UNG activity and DNA synthesis [23][24][25]. Transcription of the UNG gene is induced late in the Gl-phase resulting in an 8-12 fold increase in the transcript level, and the pattern of UNG accumulation indicated that the gene expression is regulated mainly at the transcriptional level [12].…”

Section: Introductionmentioning

confidence: 99%

Structure of the gene for human uracil—DNA glycosylase and analysis of the promoter function

Haug¹,

Skorpen²,

Lund³

et al. 1994

FEBS Letters

The gene for human uracil-DNA glycosylase (UNG) contains 4 exons and has an approximate size of 13 kb. The promoter is very GC rich and lacks a TATA box. Nested deletions of the promoter demonstrated that two SP1 elements and a putative c-MYC element proximal to the transcription initiation region were sufficient to support some 27% of the promoter activity, while a clone that in addition contained the elements E2F/SPI/CCAAT increased expression to almost 90% of the full-length construct. A region upstream of these elements appears to exert a negative control function.

Proc. Natl. Acad. Sci. U.S.A.

“…This enhancement of repair capacity has been observed with both asynchronous and synchronous cell populations, using various protocols to quantitate DNA repair. The in vitro quantitation of individual DNA repair enzyme activities during cell proliferation demonstrated that increases in the specific activities of the base excision repair enzymes uracil DNA glycosylase (8)(9)(10)(11)(12)(13)) and 3-methyladenine DNA glycosylase (12) as well as an increase in the specific activity of the 06-methylguanine methyltransferase (14) were dependent on the proliferative state of the cell. These increases in enzyme activity, in the absence of cellular insult, indicated that the induction of repair enzymes was a normal regulatory event during cell proliferation (15,16).…”

mentioning

confidence: 99%

Isolation and characterization of monoclonal antibodies directed against the DNA repair enzyme uracil DNA glycosylase from human placenta.

Arenaz¹,

Sirover²

1983

A series of monoclonal antibodies has been prepared against the base excision repair enzyme uracit DNA glycosylase isolated from human placenta. Spleen cells from BALB/ c mice immunized with purified human placentaluracil DNA glycosylase were fused with either P3X63 Ag8.653 or SP2/0 myeloma cells. Hybridomas producing antibodies directed against the placental glycosylase were identified in an enzyme-linked immunosorbent assay. Each positive hybridoma was cloned twice by limit dilution and tested for anti-glycosylase activity in an enzyme immunoprecipitation assay. Each of the four clones examined in detail precipitated enzyme activity in an immunoprecipitation reaction only in the presence of rabbit anti-mouse IgG as a second antibody. No anti-uracil DNA glycosylase activity was observed in a spontaneous hybridoma used as a control. Each monoclonal antibody immunoprecipitated uracil DNA glycosylases isolated from several human tissues. Partial crossreactivity was observed with rat liver glycosylase and with a hamster enzyme. In contrast, no crossreactivity was observed with yeast or Escherichia coli glycosylase. Glycerol gradient sedimentation analysis demonstrated that one of the antibodies bound to the glycosylase at a site that did not diminish its catalytic activity. A second monoclonal antibody bound at a determinant that affected catalytic activity. Analysis of antibody-glycosylase interactions suggests that human cells contain antigenically distinct glycosylase species that may be encoded by individual uracil DNA glycosylase genes. The potential use of these monoclonal antibodies in studies examining the regulation of.glycosylase isoenzymes during cell proliferation in normal human cells and in cells from cancer-prone individuals is considered.Recent studies have indicated that there is a correlation between the proliferative state of a eukarvotic cell and the capacity of that cell to repair DNA after chemical or physical insult. Increases in the.repair capacity of proliferating cells above basal levels present in quiescent cells have been observed for all DNA repair pathways that have been examined. These have included nueleotide excision repair after UV irradiation (1-6) or after exposure to N-acetoxyacetylaminofluorene (2, 6), base excision repair after exposure to methyl methanesulfonate (5, 6) or to sodium bisulfite (5), and repair after exposure to ionizing radiation (7). This enhancement of repair capacity has been observed with both asynchronous and synchronous cell populations, using various protocols to quantitate DNA repair. The in vitro quantitation of individual DNA repair enzyme activities during cell proliferation demonstrated that increases in the spe-