Human Uracil-DNA Glycosylase Gene: Sequence Organization, Methylation Pattern, and Mapping to Chromosome 12q23–q24.1

Haug, Terje; Skorpen, Frank; Kvaløy, Kirsti; Eftedal, Ingrid; Lund, Henning; Krokan, Hans E.

doi:10.1006/geno.1996.0485

Cited by 40 publications

(17 citation statements)

References 5 publications

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“…Alternatively, the Dnmt1 protein or the resulting methylation may actually protect against the proposed enzyme-mediated mutagenesis. It should be noted that the missense mutation assays were conducted in a wild-type background of uracil and thymine DNA glycosylase genes (29,30,58,70). This would severely limit our ability to detect enzyme-mediated and spontaneous deamination events.…”

Section: Discussionmentioning

confidence: 99%

Reduced Rates of Gene Loss, Gene Silencing, and Gene Mutation in Dnmt1-Deficient Embryonic Stem Cells

Chan¹,

Amerongen²,

Nijjar³

et al. 2001

Molecular and Cellular Biology

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Tumor suppressor gene inactivation is a crucial event in oncogenesis. Gene inactivation mechanisms include events resulting in loss of heterozygosity (LOH), gene mutation, and transcriptional silencing. The contribution of each of these different pathways varies among tumor suppressor genes and by cancer type. The factors that influence the relative utilization of gene inactivation pathways are poorly understood. In this study, we describe a detailed quantitative analysis of the three major gene inactivation mechanisms for a model gene at two different genomic integration sites in mouse embryonic stem (ES) cells. In addition, we targeted the major DNA methyltransferase gene, Dnmt1, to investigate the relative contribution of DNA methylation to these various competing gene inactivation pathways. Our data show that gene loss is the predominant mode of inactivation of a herpes simplex virus thymidine kinase neomycin phosphotransferase reporter gene (HSVTKNeo) at the two integration sites tested and that this event is significantly reduced in Dnmt1-deficient cells. Gene silencing by promoter methylation requires Dnmt1, suggesting that the expression of Dnmt3a and Dnmt3b alone in ES cells is insufficient to achieve effective gene silencing. We used a novel assay to show that missense mutation rates are also substantially reduced in Dnmt1-deficient cells. This is the first direct demonstration that DNA methylation affects point mutation rates in mammalian cells. Surprisingly, the fraction of CpG transition mutations was not reduced in Dnmt1-deficient cells. Finally, we show that methyl group-deficient growth conditions do not cause an increase in missense mutation rates in Dnmt1-proficient cells, as predicted by methyltransferase-mediated mutagenesis models. We conclude that Dnmt1 deficiency and the accompanying genomic DNA hypomethylation result in a reduction of three major pathways of gene inactivation in our model system.

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Section: Discussionmentioning

confidence: 99%

Reduced Rates of Gene Loss, Gene Silencing, and Gene Mutation in Dnmt1-Deficient Embryonic Stem Cells

Chan¹,

Amerongen²,

Nijjar³

et al. 2001

Molecular and Cellular Biology

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“…Previously, RPA has been shown to be involved in the damage recognition step of nucleotide excision repair (14), in recombination repair (15), and in replication (13), but a possible involvement in base excision repair has not been reported, except for a possible role of RPA as a transcription factor in the regulation of expression of DNA repair genes (11). A putative element for binding of RPA is located in an inhibitory region of the UNG promoter (9,10). In nucleotide excision repair, XPA interacts with both RPA2 and RPA1, but whereas RPA1 is essential for cell survival after UV light exposure, RPA2 has a more modest although significant effect (16).…”

Section: Discussionmentioning

confidence: 99%

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

Nagelhus

Haug

Singh³

et al. 1997

Journal of Biological Chemistry

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137

124

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Uracil-DNA glycosylase releases free uracil from DNA and initiates base excision repair for removal of this potentially mutagenic DNA lesion. Using the yeast twohybrid 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. Uracil-DNA glycosylase (UDG)1 is the first enzyme in base excision repair for removal of uracil from DNA and its main function is probably to remove mutagenic uracil residues resulting from deamination of cytosine in DNA (1). The subsequent steps in the base excision repair pathway include, as the minimal enzymatic requirement in vitro, an apurinic/apyrimidinic endonuclease, a deoxyribophosphodiesterase activity (which may be contributed by DNA polymerase ␤), DNA polymerase ␤, and a DNA ligase (2). In analogy to the complexity of the nucleotide excision repair pathway, base excision repair is likely to be more complex in vivo. This is in fact supported by the finding of an alternative, short patch pathway, requiring proliferating cell nuclear antigen and DNA polymerase ␦ (3, 4). A catalytically fully active form of human UDG has been expressed in Escherichia coli (5) and structure-function relationships determined by site-directed mutagenesis and x-ray crystallography (6). These studies identified this form of human UDG as a one domain structure with a positively charged DNA-binding groove. UDGs are relatively small monomeric enzymes that, at least in vitro, do not require cofactors. However, UDG is preferentially associated with replicating SV40 minichromosomes, indicating a possible interaction with components of the replication machinery (7). The gene encoding the major human UDG, UNG, is transcribed predominantly late in the G 1 -phase, resulting in a 2-3-fold increase in UDG activity early in the S-phase (8). The cell cycle regulation is consistent with the presence of several putative regulatory elements detected in the UNG gene (9), including a putative element for binding of replication protein A (RPA) (10) reported previously in D...

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“…UNG spans approx. 13.5 kb, comprises seven exons, and was assigned to chromosome 12q23-q24.1 by radiation hybrid mapping [43,59]. The promoter region and exons 1A and 1 constitute a CpG island.…”

Section: Udgs Belong To a Highly Conserved Ancient Familymentioning

confidence: 99%

DNA glycosylases in the base excision repair of DNA

Krokan

Standal

Slupphaug

1997

Biochemical Journal

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766

566

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A wide range of cytotoxic and mutagenic DNA bases are removed by different DNA glycosylases, which initiate the base excision repair pathway. DNA glycosylases cleave the Nglycosylic bond between the target base and deoxyribose, thus releasing a free base and leaving an apurinic\apyrimidinic (AP) site. In addition, several DNA glycosylases are bifunctional, since they also display a lyase activity that cleaves the phosphodiester backbone 3h to the AP site generated by the glycosylase activity. Structural data and sequence comparisons have identified common features among many of the DNA glycosylases. Their active sites have a structure that can only bind extrahelical target bases, as observed in the crystal structure of human uracil-DNA glycosylase in a complex with double-stranded DNA. Nucleotide flipping is apparently actively facilitated by the enzyme. With bacteriophage T4 endonuclease V, a pyrimidine-

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Human Uracil-DNA Glycosylase Gene: Sequence Organization, Methylation Pattern, and Mapping to Chromosome 12q23–q24.1

Cited by 40 publications

References 5 publications

Reduced Rates of Gene Loss, Gene Silencing, and Gene Mutation in Dnmt1-Deficient Embryonic Stem Cells

Reduced Rates of Gene Loss, Gene Silencing, and Gene Mutation in Dnmt1-Deficient Embryonic Stem Cells

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

DNA glycosylases in the base excision repair of DNA

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