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

Haug, Terje; Skorpen, Frank; Lund, Henning; Krokan, Hans E.

doi:10.1016/0014-5793(94)01042-0

Cited by 23 publications

(9 citation statements)

References 51 publications

(42 reference statements)

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“…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 DNA repair genes in yeast (11). RPA is a trimeric protein required for initiation of DNA replication (12,13), in the initial steps of nucleotide excision repair in physical interaction with XPA (14), as well as in recombination repair (15).…”

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confidence: 59%

“…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%

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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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confidence: 59%

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

Self Cite

137

124

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“…UDG fold and residue function mapped onto the structure‐based alignment of UDG sequences (DDBJ/EMBL/GenBank accession numbers in parentheses) are from: (1) Homo sapiens (Haug et al ., 1994) (1296803); (2) Mus musculus (Svendsen et al ., 1997) (1762318); (3) E.coli (Varshney et al ., 1988) (148149); (4) human herpesvirus 1 (HSV‐1) (McGeoch et al ., 1988) (221724); (5) human cytomegalovirus (Chee et al ., 1990) (1780896); and (6) vaccinia virus (Goebel et al ., 1990) (137597). Secondary structure assignments, indicated above the sequence alignment, refer to the current co‐crystal structures of human UDG in complexes with DNA, and do not include the two small β‐strands (Mol et al ., 1995).…”

Section: Resultsmentioning

confidence: 99%

Base excision repair initiation revealed by crystal structures and binding kinetics of human uracil-DNA glycosylase with DNA

et al. 1998

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Three high-resolution crystal structures of DNA complexes with wild-type and mutant human uracil-DNA glycosylase (UDG), coupled kinetic characterizations and comparisons with the refined unbound UDG structure help resolve fundamental issues in the initiation of DNA base excision repair (BER): damage detection, nucleotide flipping versus extrahelical nucleotide capture, avoidance of apurinic/apyrimidinic (AP) site toxicity and coupling of damage-specific and damagegeneral BER steps. Structural and kinetic results suggest that UDG binds, kinks and compresses the DNA backbone with a 'Ser-Pro pinch' and scans the minor groove for damage. Concerted shifts in UDG simultaneously form the catalytically competent active site and induce further compression and kinking of the double-stranded DNA backbone only at uracil and AP sites, where these nucleotides can flip at the phosphatesugar junction into a complementary specificity pocket. Unexpectedly, UDG binds to AP sites more tightly and more rapidly than to uracil-containing DNA, and thus may protect cells sterically from AP site toxicity. Furthermore, AP-endonuclease, which catalyzes the first damage-general step of BER, enhances UDG activity, most likely by inducing UDG release via shared minor groove contacts and flipped AP site binding. Thus, AP site binding may couple damage-specific and damage-general steps of BER without requiring direct protein-protein interactions.

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“…Mutational studies have indicated that the c-Myc element is a positive regulator of UDG expression, whereas the E2F element (and overexpression of E2F) regulates UDG expression negatively. In addition, Sp1 elements are present in both the P A and the P B promoters, and are required for effective expression ( [80] ; T. Haug, P. A. Aas, F. Skorpen, V. Malm, C. Skjeldbred and H. E. Krokan, unpublished work). The nuclear and mitochondrial forms of UDG are probably not equally regulated, since their promoters (P A and P B ) are structurally different.…”

Section: Regulation Of Udg Expressionmentioning

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

Cited by 23 publications

References 51 publications

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

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

Base excision repair initiation revealed by crystal structures and binding kinetics of human uracil-DNA glycosylase with DNA

DNA glycosylases in the base excision repair of DNA

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