DNA bending and a flip-out mechanism for base excision by the helix–hairpin–helix DNA glycosylase, Escherichia coli AlkA

Hollis, Thomas; Ichikawa, Yoshitaka; Ellenberger, Tom

doi:10.1093/emboj/19.4.758

Cited by 203 publications

(289 citation statements)

References 43 publications

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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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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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“…Although the overall identity between the predicted protein product of hp0585 and E. coli Nth is low, it is possible to identify in the putative HpNth the pattern of cysteines giving rise to the iron-sulfur cluster and the helix-hairpin-helix motif, characteristic of the EndoIII family of proteins (27,28). To examine whether H. pylori hp0585 codes for a protein with EndoIII activity, hp585A or hp585T fused to GST were expressed in E. coli SW2-38.…”

Section: Expression Of H Pylori Hp0585 Complements the E Coli Nth Nmentioning

confidence: 99%

Pathogen DNA as target for host-generated oxidative stress: Role for repair of bacterial DNA damage in Helicobacter pylori colonization

O’Rourke

Chevalier

Pinto

et al. 2003

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

122

110

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Helicobacter pylori elicits an oxidative stress during host colonization. This oxidative stress is known to cause lesions in the host DNA. Here we addressed the question as to whether the pathogen DNA is subject to lethal or mutational damage by the host-generated oxidative response. H. pylori Hpnth mutants unable to repair oxidized pyrimidines from the bacterial DNA were generated. H. pylori strains lacking a functional endonuclease III (HpNth) showed elevated spontaneous and induced mutation rates and were more sensitive than the parental strain to killing by exposure to oxidative agents or activated macrophages. Although under laboratory conditions the Hpnth mutant strain grows as well as the wild-type strain, in a mouse infection the stomach bacterial load gradually decreases while the population in the wild-type strain remains stable, showing that endonuclease III deficiency reduces the colonization capacity of the pathogen. In coinfection experiments with a wild-type strain, Hpnth cells are eradicated 15 days postinfection (p.i.) even when inoculated in a 1:9 wild-type:mutant strain ratio, revealing mutagenic lesions that are counterselected under competition conditions. These results show that the host effectively induces lethal and premutagenic oxidative DNA adducts on the H. pylori genome. The possible consequences of these DNA lesions on the adaptability of H. pylori strains to new hosts are discussed.

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“…It should be clearly pointed out that the cleavage intensities of U 6 cannot be directly compared with T 6 in these experiments, because free uridine is about 5-fold less reactive than free thymidine to oxidation by KMnO 4 (28). Therefore, the sensitivities of U 6 or T 6 in the duplex context must be compared with the corresponding bases in the single-stranded form as we have done in Fig. 2B.…”

Section: Design and Characterization Of Normal And Pyrene Wedgementioning

confidence: 99%

“…1A), and several enzyme hydrogen bond donors and acceptors pull and stabilize the extrahelical uracil in the active site. The pinch-push-pull mechanism appears to represent a highly conserved mechanism to promote base flipping, because corresponding interactions have been found in the structures of all DNA glycosylase-DNA complexes (6,7).…”

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confidence: 99%

Turning On Uracil-DNA Glycosylase Using a Pyrene Nucleotide Switch

Jiang

Kwon

Stivers

2001

Journal of Biological Chemistry

105

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Base flipping is a highly conserved process by which enzymes swivel an entire nucleotide from the DNA base stack into their active site pockets. Uracil DNA glycosylase (UDG) is a paradigm enzyme that uses a base flipping mechanism to catalyze the hydrolysis of the Nglycosidic bond of 2-deoxyuridine (2-dUrd) in DNA as the first step in uracil base excision repair. Flipping of 2-dUrd by UDG has been proposed to follow a "pushing" mechanism in which a completely conserved leucine side chain (Leu-191) is inserted into the DNA minor groove to expel the uracil. Here we report a novel implementation of the "chemical rescue" approach to show that the weak binding affinity and low catalytic activity of L191A or L191G can be completely or partially restored by substitution of a pyrene (Y) nucleotide wedge on the DNA strand opposite to the uracil base (U/A to U/Y). These results indicate that pyrene acts both as a wedge to push the uracil from the base stack in the free DNA and as a "plug" to hinder its reinsertion after base flipping. Pyrene rescue should serve as a useful and novel tool to diagnose the functional roles of other amino acid side chains involved in base flipping.

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DNA bending and a flip-out mechanism for base excision by the helix–hairpin–helix DNA glycosylase, Escherichia coli AlkA

Cited by 203 publications

References 43 publications

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

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

Pathogen DNA as target for host-generated oxidative stress: Role for repair of bacterial DNA damage in Helicobacter pylori colonization

Turning On Uracil-DNA Glycosylase Using a Pyrene Nucleotide Switch

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