Properties of a Recombinant Human Uracil-DNA Glycosylase from the UNG Gene and Evidence that UNG Encodes the Major Uracil-DNA Glycosylase

Slupphaug, Geir; Eftedal, Ingrid; Kavli, Bodil; Bharati, Sangeeta; Helle, Nils M.; Haug, Terje; Levine, David; Krokan, Hans E.

doi:10.1021/bi00001a016

Cited by 253 publications

(270 citation statements)

References 49 publications

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“…The rate differs as much as 20-fold between the ' best ' (A\T\UAA) and ' worst ' (G\CUG\C\T) sequences. Usually, but not always [34], the rate of removal is greater from U\G mispairs than from U :A pairs [35][36][37]. A low removal rate tends to be correlated with the occurrence of ' hot spots ' for mutation [36], similar to the correlation between slow spots for the repair of cyclobutyl dimers and hot spots for mutations [38,39].…”

Section: Udgsmentioning

confidence: 91%

“…Ugi forms an essentially irreversible complex with UDG [68,69], and the specificity of the interaction [70,71] indicates that only structurally related UDGs will bind to Ugi. Furthermore, antibodies raised against homogeneous human UDG detected both mitochondrial and nuclear forms, indicating that these forms are closely related [37,64,72].…”

Section: Similarities Between the Nuclear And Mitochondrial Forms Of Udgmentioning

confidence: 95%

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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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Section: Udgsmentioning

confidence: 91%

Section: Similarities Between the Nuclear And Mitochondrial Forms Of Udgmentioning

confidence: 95%

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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“…The core catalytic domain of UNG (UNGΔ84), which lacks the N-terminal 84 amino acids of UNG2 [5], was overproduced in E. coli and purified as described by Chen et al [35]. PCNA LC59 was overproduced in E. coli BL21 (DE3) and purified as described previously for wild-type PCNA, except that the LC59 double mutant flowed through, rather than binding to, the phenyl-Sepharose column [32].…”

Section: Protein Purificationmentioning

confidence: 99%

“…required for subcellular sorting, while the core catalytic domain (UNGΔ84) is common to the two forms [3,4]. All forms of UNG examined remove uracil opposite A or G in doublestranded DNA as well as from single-stranded DNA [3,5]. At least four different human nuclear DNA glycosylases, UNG2, single-strand selective monofunctional uracil-DNA glycosylase (SMUG1), thymine-DNA glycosylase (TDG) and methyl-CpG binding domain protein 4 (MBD4, also known as MED1) have been reported to removal uracil from U·G mispairs [6][7][8], and it has not yet been determined whether the different uracil-DNA glycosylases have distinct or overlapping physiological functions.…”

Section: Introductionmentioning

confidence: 99%

Physical and functional interaction of human nuclear uracil-DNA glycosylase with proliferating cell nuclear antigen

Bennett

2005

DNA Repair

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Uracil residues arise in DNA by the misincorporation of dUMP in place of dTMP during DNA replication or by the deamination of cytosine in DNA. Uracil-DNA glycosylase initiates DNA base excision repair of uracil residues by catalyzing the hydrolysis of the N-glycosylic bond linking the uracil base to deoxyribose. In human cells, the nuclear form of uracil-DNA glycosylase (UNG2) contains a conserved PCNA-binding motif located at the N-terminus that has been implicated experimentally in binding PCNA. Here we use purified preparations of UNG2 and PCNA to demonstrate that UNG2 physically associates with PCNA. UNG2 co-eluted with PCNA during size exclusion chromatography and bound to a PCNA affinity column. Association of UNG2 with PCNA was abolished by the addition of 100 mM NaCl, and significantly decreased in the presence of 10 mM MgCl 2 . The functional significance of the UNG2·PCNA association was demonstrated by UNG2 activity assays. Addition of PCNA (30-810 pmol) to standard uracil-DNA glycosylase reactions containing linear [uracil-3 H]DNA stimulated UNG2 catalytic activity up to 2.6-fold. UNG2 activity was also stimulated by 7.5 mM MgCl 2 . The stimulatory effect of PCNA was increased by the addition of MgCl 2 ; however, the dependence on PCNA concentration was the same, indicating that the effects of MgCl 2 and PCNA on UNG2 activity occurred by independent mechanisms. Loading of PCNA onto the DNA substrate was required for stimulation, as the activity of UNG2 on circular DNA substrates was not affected by the addition of PCNA. Addition of replication factor C and ATP to reactions containing 90 pmol of PCNA resulted in two-fold stimulation of UNG2 activity on circular DNA.

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“…UNG proteins are relatively small monomeric proteins that usually do not require cofactors or ions for their activity. They are also highly specific against uracil in both single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA), with a preference for ssDNA (64,75).…”

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confidence: 99%

Characterization of the Uracil-DNA Glycosylase Activity of Epstein-Barr Virus BKRF3 and Its Role in Lytic Viral DNA Replication

Huang

Wang

et al. 2007

J Virol

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Uracil-DNA glycosylases (UDGs) of the uracil-N-glycosylase (UNG) family are the primary DNA repair enzymes responsible for removal of inappropriate uracil from DNA. Recent studies further suggest that the nuclear human UNG2 and the UDGs of large DNA viruses may coordinate with their DNA polymerase accessory factors to enhance DNA replication. Based on its amino acid sequence, the putative UDG of Epstein-Barr virus (EBV), BKRF3, belongs to the UNG family of proteins, and it was demonstrated previously to enhance oriLyt-dependent DNA replication in a cotransfection replication assay. However, the expression and enzyme activity of EBV BKRF3 have not yet been characterized. In this study, His-BKRF3 was expressed in bacteria and purified for biochemical analysis. Similar to the case for the Escherichia coli and human UNG enzymes, His-BKRF3 excised uracil from single-stranded DNA more efficiently than from double-stranded DNA and was inhibited by the purified bacteriophage PBS1 inhibitor Ugi. In addition, BKRF3 was able to complement an E. coli ung mutant in rifampin and nalidixic acid resistance mutator assays. The expression kinetics and subcellular localization of BKRF3 products were detected in EBV-positive lymphoid and epithelial cells by using BKRF3-specific mouse antibodies. Expression of BKRF3 is regulated mainly by the immediateearly transcription activator Rta. The efficiency of EBV lytic DNA replication was slightly affected by BKRF3 small interfering RNA (siRNA), whereas cellular UNG2 siRNA or inhibition of cellular and viral UNG activities by expressing Ugi repressed EBV lytic DNA replication. Taking these results together, we demonstrate the UNG activity of BKRF3 in vitro and in vivo and suggest that UNGs may participate in DNA replication or repair and thereby promote efficient production of viral DNA.

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Properties of a Recombinant Human Uracil-DNA Glycosylase from the UNG Gene and Evidence that UNG Encodes the Major Uracil-DNA Glycosylase

Cited by 253 publications

References 49 publications

DNA glycosylases in the base excision repair of DNA

DNA glycosylases in the base excision repair of DNA

Physical and functional interaction of human nuclear uracil-DNA glycosylase with proliferating cell nuclear antigen

Characterization of the Uracil-DNA Glycosylase Activity of Epstein-Barr Virus BKRF3 and Its Role in Lytic Viral DNA Replication

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