Crystal structure of Escherichia coli uracil DNA glycosylase and its complexes with uracil and glycerol: Structure and glycosylase mechanism revisited

Xiao, Gaoyi; Tordova, Maria; Jagadeesh, Jaya; Drohat, Alexander C.; Stivers, James T.; Gilliland, Gary L.

doi:10.1002/(sici)1097-0134(19990401)35:1<13::aid-prot2>3.3.co;2-u

Cited by 30 publications

(44 citation statements)

References 21 publications

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“…Furthermore, combined loss of p53 function and DNA double-stranded break responses such as ataxiatelangiectasia mutated (ATM) or the nonhomologous endjoining pathway would lead to the accelerated appearance of VOL. 81,2007 EBV URACIL-DNA GLYCOSYLASE 1205…”

Section: Discussionmentioning

confidence: 57%

“…The efficiency of plating showed that E. coli growth was not influ-VOL. 81,2007 EBV URACIL- DNA GLYCOSYLASE 1197 enced by the expression of BKRF3, BKRF3mt, or hUNG (Fig. 2B).…”

Section: Resultsmentioning

confidence: 99%

“…Although these share a common structural fold, they are surprisingly divergent at the amino acid level (1,51). The uracil-N-glycosylase (UNG) family (family 1) of proteins are the most ubiquitous, share similar biochemical properties, and are highly conserved at both the amino acid and structural levels (61,65,81). UNG proteins are relatively small monomeric proteins that usually do not require cofactors or ions for their activity.…”

mentioning

confidence: 99%

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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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Section: Discussionmentioning

confidence: 57%

“…The efficiency of plating showed that E. coli growth was not influ-VOL. 81,2007 EBV URACIL- DNA GLYCOSYLASE 1197 enced by the expression of BKRF3, BKRF3mt, or hUNG (Fig. 2B).…”

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

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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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“…Within this family, hUNG shares almost 60% amino acid identity with E. coli, whereas it drops to less than 20% with VACV UNG (5). Although the UNG overall structures are very similar (1.0 Å rms deviation with 207 aligned residues) when E. coli (Protein Data Bank entry 3EUG) (34) and hUNG (Protein Data Bank entry 1AKZ) (25) are aligned, D4 from His-D4⅐A20 1-50 ⅐DNA shows an rms deviation of 2.3 Å when compared with the different structures of hUNG bound to DNA (22)(23)(24).…”

Section: Discussionmentioning

confidence: 96%

Crystal Structure of the Vaccinia Virus Uracil-DNA Glycosylase in Complex with DNA

Burmeister¹,

Tarbouriech²,

Fender³

et al. 2015

Journal of Biological Chemistry

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Background: D4 is a uracil-DNA glycosylase (UNG) and an essential component of the vaccinia virus DNA polymerase holoenzyme. Results: The crystal structure of D4 in complex with DNA is presented. Conclusion: The D4⅐DNA contacts exhibit major differences compared with the human UNG⅐DNA complex. Significance: This work allows a better understanding of the structural determinants required for UNG function.

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“…This would also be more consistent with the structural and mutagenesis data that have been reported for flipped out bases, which are found in close proximity to just one aromatic amino acid residue. This is a tryptophan in the case of the E. coli repair enzyme AlkA (26) and the Tn5 transposon (27), a tyrosine for human 3-methyladenine DNA glycosylase and B. subtilis DNA polymerase I (25,28), and a phenylalanine for N6-adenine DNA methyltransferase (29) and E. coli and human uracil DNA glycosylase (30,31). Another aromatic residue could be involved in the above mentioned forcing out of the flipped base or could be interacting edge-on with it, like tyrosines 162 and 159, respectively, of human 3-methyladenine DNA glycosylase (25) …”

Section: Discussionmentioning

confidence: 99%

Different Roles for Basic and Aromatic Amino Acids in Conserved Region 2 of Escherichia coli ς70 in the Nucleation and Maintenance of the Single-stranded DNA Bubble in Open RNA Polymerase-Promoter Complexes

Tomsic

Tsujikawa

Panaghie

et al. 2001

Journal of Biological Chemistry

122

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Amino acid residues in region 2 of 70 have been shown to play an important role in the strand separation step that is necessary for formation of the functional or open RNA polymerase-promoter complex. Here we present a comparison of the roles of basic and aromatic amino acids in the accomplishment of this process, using RNA polymerase bearing alanine substitutions for both types of amino acids in region 2. We determined the effects of the substitutions on the kinetics of open complex formation, as well as on the ability of the RNA polymerase to form complexes with singlestranded DNA, and with forked DNA duplexes carrying a single-stranded overhang consisting of bases in the ؊10 region. We concluded that two basic amino acids (Lys 414 and Lys 418 ) are important for promoter binding and demonstrated distinct roles, at a subsequent step, for two aromatic amino acids (Tyr 430 and Trp 433 ). It is likely that these four amino acids, which are close to each other in the structure of 70 , together are involved in the nucleation of the strand separation process.RNA synthesis in prokaryotes is carried out by a multisubunit RNA polymerase commonly referred to as the core enzyme (E).1 For promoter recognition, a sigma (initiation) factor is required; it interacts with the core polymerase to yield the holoenzyme (E), which is able to form an initiation-competent complex at promoter sequences in a multistep process involving conformational changes in both the protein and the DNA (1-3). A striking feature of such a complex is a region of strand separation that spans about 14 base pairs from the upstream edge of the conserved Ϫ10 promoter element to just beyond the start site of transcription initiation (4). It is thought that, kinetically, strand separation initiates in the Ϫ10 region and proceeds in a downstream direction. Measurement of the size and location of the region of strand separation as a function of temperature shows that at low temperatures a small single-stranded region can be detected that, as the temperature is increased, expands toward the start site (3, 5-7). In addition, the introduction of nicks and mismatches in the Ϫ10 region is more effective in the acceleration of open complex formation than if such distortions are introduced at a more downstream position (8, 9).The predominant sigma factor in Escherichia coli, which enables recognition of promoters of housekeeping genes, is referred to as 70 . Sequence comparison has shown that a large group of sigma factors shows significant homology to 70 . Four regions of sequence conservation have been identified, of which some have been subdivided to reflect the most extensive sequence conservation (10). A large body of data has implicated region 2.3 of the main sigma factors of E. coli, Bacillus subtilis, and other prokaryotes in the nucleation of the strand separation. This process eventually results in the formation of the active or open complex, possibly by facilitating base flipping of the highly conserved A at Ϫ11 of the nontemplate strand. The support...

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Crystal structure of Escherichia coli uracil DNA glycosylase and its complexes with uracil and glycerol: Structure and glycosylase mechanism revisited

Cited by 30 publications

References 21 publications

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

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

Crystal Structure of the Vaccinia Virus Uracil-DNA Glycosylase in Complex with DNA

Different Roles for Basic and Aromatic Amino Acids in Conserved Region 2 of Escherichia coli ς70 in the Nucleation and Maintenance of the Single-stranded DNA Bubble in Open RNA Polymerase-Promoter Complexes

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