Functions of base flipping in E. coli nucleotide excision repair

Malta, Erik; Verhagen, Carlo P.; Moolenaar, Geri F.; Filippov, Dmitri V.; Marel, Gijs A. van der; Goosen, Nora

doi:10.1016/j.dnarep.2008.06.011

Cited by 21 publications

(22 citation statements)

References 25 publications

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“…The enhancement of 2-AP fluorescence induced by DNAbinding proteins has been interpreted previously as evidence for DNA strand separation and/or base flipped out of its helical conformation. [42][43][44][45] Although the precise mechanism remains obscure, helical distortions may account for the fluorescence enhancement induced by PI-MleI. We conclude, however, that changes in DNA conformation are apparent at the cleavage sites upon binding of PI-MleI and its variants; the absence of such distortion at the binding DNA sequence (Fig.…”

Section: Dna-binding Analysis Of Pi-mlei and Its Variants By Electropmentioning

confidence: 72%

“…41 Fluorescent base analogs, such as 2-AP, are often used to monitor such conformational changes involving proteinnucleic acid interaction. [42][43][44][45] To gain insights into the functional differences in the target DNA selectivity of wild-type PI-MleI and its variants, we used 2-AP fluorescence to monitor changes induced in double-helical DNA by these enzymes. We observed that the fluorescence intensities induced by PI-MleI and its variants with the target DNA substrate at the cleavage site followed a hierarchical manner in the following order: wild-type PI-MleI : D122C : D122T : D218A > D122A > D193A (Fig.…”

Section: Dna-binding Analysis Of Pi-mlei and Its Variants By Electropmentioning

confidence: 99%

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Mutational analysis of active‐site residues in the Mycobacterium leprae RecA intein, a LAGLIDADG homing endonuclease: Asp¹²² and Asp¹⁹³ are crucial to the double‐stranded DNA cleavage activity whereas Asp²¹⁸ is not

2009

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Mycobacterium leprae recA harbors an in-frame insertion sequence that encodes an intein homing endonuclease (PI-MleI). Most inteins (intein endonucleases) possess two conserved LAGLIDADG (DOD) motifs at their active center. A common feature of LAGLIDADG-type homing endonucleases is that they recognize and cleave the same or very similar DNA sequences. However, PI-MleI is distinctive from other members of the family of LAGLIDADG-type HEases for its modular structure with functionally separable domains for DNA-binding and cleavage, each with distinct sequence preferences. Sequence alignment analyses of PI-MleI revealed three putative LAGLIDADG motifs; however, there is conflicting bioinformatics data in regard to their identity and specific location within the intein polypeptide. To resolve this conflict and to determine the activesite residues essential for DNA target site recognition and double-stranded DNA cleavage, we performed site-directed mutagenesis of presumptive catalytic residues in the LAGLIDADG motifs. Analysis of target DNA recognition and kinetic parameters of the wild-type PI-MleI and its variants disclosed that the two amino acid residues, Asp 122 (in Block C) and Asp 193 (in functional Block E), are crucial to the double-stranded DNA endonuclease activity, whereas Asp 218 (in pseudo-Block E) is not. However, despite the reduced catalytic activity, the PI-MleI variants, like the wild-type PIMleI, generated a footprint of the same length around the insertion site. The D122T variant showed significantly reduced catalytic activity, and D122A and D193A mutations although failed to affect their DNA-binding affinities, but abolished the double-stranded DNA cleavage activity. On the other hand, D122C variant showed approximately twofold higher double-stranded DNA cleavage activity, compared with the wild-type PI-MleI. These results provide compelling evidence that Asp 122 and Additional Supporting Information may be found in the online version of this article.Abbreviations: 2-AP, 2 aminopurine; bp, base pair; BPB, bromophenol blue; EDTA, ethylene diamine tetraacetic acid; form I DNA, negatively supercoiled DNA; form II DNA, nicked circular DNA; form III DNA, linear double-stranded DNA; ODN, oligonucleotide; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; PI-MleI, M. leprae RecA intein; SDS, sodium dodecyl sulfate. Asp 193 in DOD motif I and II, respectively, are bona fide active-site residues essential for DNA cleavage activity. The implications of these results are discussed in this report.

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Section: Dna-binding Analysis Of Pi-mlei and Its Variants By Electropmentioning

confidence: 72%

Section: Dna-binding Analysis Of Pi-mlei and Its Variants By Electropmentioning

confidence: 99%

Mutational analysis of active‐site residues in the Mycobacterium leprae RecA intein, a LAGLIDADG homing endonuclease: Asp¹²² and Asp¹⁹³ are crucial to the double‐stranded DNA cleavage activity whereas Asp²¹⁸ is not

2009

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“…In the most extreme case, the lesion may be completely blocked at the tunnel entrance as in the “steric gate” model (27). A recent fluorescence study suggests the possibility that the nature of the lesion determines how far the damaged strand can translocate behind the β-hairpin (22). A cholesterol lesion linked directly to the DNA backbone is suggested to be translocated further behind the β-hairpin as compared to a menthol lesion which is directly linked to a base.…”

Section: Discussionmentioning

confidence: 99%

“…Further fluorescence studies have recently shown that this flipped-out base helps stabilize the UvrB/DNA binding complex (22). In addition, studies with UvrB mutants have shown that stacking interactions occur between UvrB tyrosine residues and several flipped out bases around the damaged base, including one on the damaged strand and one on the partner strand (23-25).…”

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confidence: 99%

Exploring Damage Recognition Models in Prokaryotic Nucleotide Excision Repair with a Benzo[a]pyrene-Derived Lesion in UvrB

Jia¹,

Kropachev²,

Ding³

et al. 2009

Biochemistry

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The UvrB protein is a central unit for damage recognition in the prokaryotic nucleotide excision repair system, which excises bulky DNA lesions. We have utilized molecular modeling and MD simulations based on crystal structures, mutagenesis, and fluorescence data, to model the 10R-(+)- [a]pyrene, at different locations on the inner and outer strand in UvrB. Our results suggest that this lesion is accommodated on the inner strand where it might translocate through the tunnel created by the β-hairpin and the UvrB domain 1B, and ultimately could be housed in the pocket behind the β-hairpin prior to excision by UvrC. Lesions that vary in size and shape may be stopped at the gate to the tunnel, within the tunnel or in the pocket when UvrC initiates excision. Common features of β-hairpin intrusion between the two DNA strands and nucleotide flipping manifested in structures of prokaryotic and eukaryotic NER lesion recognition proteins are consistent with common recognition mechanisms, based on lesion-induced local thermodynamic distortion/destabilization and nucleotide flipping. cis-anti-B[a]P-N 2 -dG lesion, derived from the tumorigenic (+) anti-B[a]PDE metabolite of benzo KeywordsNucleotide Excision Repair; UvrB; Lesion Recognition; Flip Out; Lesion Translocation Supporting Information Available The molecular dynamics protocol and a translocation movie are provided. Figure S1 shows the superimposition of the ATP binding site when modeling the ATP from 1D9Z into 2FDC, and the superimposition of protein residues 420-460 of 2FDC to residues 418-458 of 2D7D to model a missing loop from 2D7D to 2FDC. Figure S2 shows plots of the all-atom RMSD of the current relative to the starting structure as a function of time for the whole UvrB/DNA complex, and for just the damaged and undamaged DNA strands. Figure S3 shows plots of the torsion angles χ, α′ and β′ for all complex models in the selected time frames for analysis. Figure S4 shows the flipping and stacking properties of the bases in representative structures of each model. Figure S5 Table S1 gives glycosidic torsion angle χ values in the initial models for MD simulations. Table S2 gives AMBER atom type, connection type, and partial charge assignments for the (+)-cis-B[a]P-N 2 -dG adduct. Table S3 gives AMBER atom type, connection type, and partial charge assignments for ATP. Table S4 gives AMBER atom type, connection type, and partial charge assignments for Mg 2+ . Table S5 gives box sizes and number of waters and counterions in the MD simulation initial models. This material is available free of charge via the Internet at http://pubs.acs.org. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2010 November 3. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptDamage to DNA occurs continuously in living organisms, either endogenously through reactive oxygen species (ROS) produced during normal metabolic processes or exogenously from sunlight, pollutants, dietary mutagens and other factors (1). To preserve th...

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“…Multiple DNA-repair enzymes have been studied with this method, including methyltransferases, 82–91 uracil DNA glycosylase, 92,93 helicase, 35 T4 endonuclease, 94 and UvrABC endonucleases. 95 …”

Section: Dna Backbone As Fluorophore Carriermentioning

confidence: 99%

Fluorescent DNA-based enzyme sensors

2011

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Fluorescent sensors that make use of DNA structures have become widely useful in monitoring enzymatic activities. Early studies focused primarily on enzymes that naturally use DNA or RNA as the substrate. However, recent advances in molecular design have enabled the development of nucleic acid sensors for a wider range of functions, including enzymes that do not normally bind DNA or RNA. Nucleic acid sensors present some potential advantages over classical small-molecule sensors, including water solubility and ease of synthesis. An overview of the multiple strategies under recent development is presented in this critical review, and expected future developments in microarrays, single molecule analysis, and in vivo sensing are discussed (160 references).

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Functions of base flipping in E. coli nucleotide excision repair

Cited by 21 publications

References 25 publications

Mutational analysis of active‐site residues in the Mycobacterium leprae RecA intein, a LAGLIDADG homing endonuclease: Asp¹²² and Asp¹⁹³ are crucial to the double‐stranded DNA cleavage activity whereas Asp²¹⁸ is not

Mutational analysis of active‐site residues in the Mycobacterium leprae RecA intein, a LAGLIDADG homing endonuclease: Asp¹²² and Asp¹⁹³ are crucial to the double‐stranded DNA cleavage activity whereas Asp²¹⁸ is not

Exploring Damage Recognition Models in Prokaryotic Nucleotide Excision Repair with a Benzo[a]pyrene-Derived Lesion in UvrB

Fluorescent DNA-based enzyme sensors

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Functions of base flipping in E. coli nucleotide excision repair

Cited by 21 publications

References 25 publications

Mutational analysis of active‐site residues in the Mycobacterium leprae RecA intein, a LAGLIDADG homing endonuclease: Asp122 and Asp193 are crucial to the double‐stranded DNA cleavage activity whereas Asp218 is not

Mutational analysis of active‐site residues in the Mycobacterium leprae RecA intein, a LAGLIDADG homing endonuclease: Asp122 and Asp193 are crucial to the double‐stranded DNA cleavage activity whereas Asp218 is not

Exploring Damage Recognition Models in Prokaryotic Nucleotide Excision Repair with a Benzo[a]pyrene-Derived Lesion in UvrB

Fluorescent DNA-based enzyme sensors

Contact Info

Product

Resources

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Mutational analysis of active‐site residues in the Mycobacterium leprae RecA intein, a LAGLIDADG homing endonuclease: Asp¹²² and Asp¹⁹³ are crucial to the double‐stranded DNA cleavage activity whereas Asp²¹⁸ is not

Mutational analysis of active‐site residues in the Mycobacterium leprae RecA intein, a LAGLIDADG homing endonuclease: Asp¹²² and Asp¹⁹³ are crucial to the double‐stranded DNA cleavage activity whereas Asp²¹⁸ is not