Uvr excision repair protein complex of Escherichia coli binds to the convex side of a cisplatin-induced kink in the DNA.

Visse, Robert; Ruijter, M de; Brouwer, Jaap; Brandsma, Jourica A.; Putte, Pieter van de

doi:10.1016/s0021-9258(20)89491-5

Cited by 59 publications

(29 citation statements)

References 28 publications

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“…Interestingly, only low amounts of drUvrA1 were needed for efficient incision activity, whereas higher concentrations of drUvrB and drUvrC were needed for optimal processing of the substrate, indicating that drUvrA1 is acting catalytically, as reported in earlier studies 12 , 13 , 40 . In the optimal reaction conditions, drUvrA1 binding to the FdT DNA substrates was very efficient, with a Kd close to 10 nM, as shown by the fluorescence-polarization measurements.…”

Section: Discussionsupporting

confidence: 66%

“…Bacterial NER has been reconstituted in vitro using the three essential proteins, UvrA, UvrB, and UvrC, and either plasmid or short DNA oligonucleotides as substrates 9 – 13 . Although several of the early studies made use of Escherichia coli Uvr proteins 9 , 12 – 21 , many of the more recent mechanistic studies of bacterial NER have relied on the use of Uvr proteins from thermophilic bacteria ( Bacillus caldotenax , Geobacillus stearothermophilus , Thermatoga maritima , and Thermus thermophilus ) 6 , 10 , 22 – 27 and, in many cases, due to solubility issues, Uvr proteins from different sources were combined to set up functional incision assays.…”

Section: Introductionmentioning

confidence: 99%

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In vitro reconstitution of an efficient nucleotide excision repair system using mesophilic enzymes from Deinococcus radiodurans

et al. 2022

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Nucleotide excision repair (NER) is a universal and versatile DNA repair pathway, capable of removing a very wide range of lesions, including UV-induced pyrimidine dimers and bulky adducts. In bacteria, NER involves the sequential action of the UvrA, UvrB and UvrC proteins to release a short 12- or 13-nucleotide DNA fragment containing the damaged site. Although bacterial NER has been the focus of numerous studies over the past 40 years, a number of key questions remain unanswered regarding the mechanisms underlying DNA damage recognition by UvrA, the handoff to UvrB and the site-specific incision by UvrC. In the present study, we have successfully reconstituted in vitro a robust NER system using the UvrABC proteins from the radiation resistant bacterium, Deinococcus radiodurans. We have investigated the influence of various parameters, including temperature, salt, protein and ATP concentrations, protein purity and metal cations, on the dual incision by UvrABC, so as to find the optimal conditions for the efficient release of the short lesion-containing oligonucleotide. This newly developed assay relying on the use of an original, doubly-labelled DNA substrate has allowed us to probe the kinetics of repair on different DNA substrates and to determine the order and precise sites of incisions on the 5′ and 3′ sides of the lesion. This new assay thus constitutes a valuable tool to further decipher the NER pathway in bacteria.

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Section: Discussionsupporting

confidence: 66%

Section: Introductionmentioning

confidence: 99%

In vitro reconstitution of an efficient nucleotide excision repair system using mesophilic enzymes from Deinococcus radiodurans

et al. 2022

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“…Presumably UvrABC is able to recognize di¡erential structural or dynamic features between Et 743^DNA adducts on the top and bottom strands. Extra 5P incisions have also been reported for cisplatin [32], where two major incision products occurred 8 and 15 bases from the site of the lesion. However, a more de¢ned system seemed necessary since the sequences shown in Fig.…”

Section: Uvrabc Incision Frequency Of Dna-containing Et 743mentioning

confidence: 89%

“…For the isolation of a 209-bp fragment labeled at the 5P-end with 32 P, the pCAT plasmid (New England Biolabs) was ¢rst digested with HindIII followed by treatment with shrimp alkaline phosphatase at 37³C for 2 h. The dephosphorylated fragment was extracted with phenol^chloroform and then ethanol-precipitated. The precipitated DNA was labeled with [Q-32 P]ATP in the presence of T4 polynucleotide kinase.…”

Section: Puri¢cation Of Uvrabc Proteinsmentioning

confidence: 99%

The inefficiency of incisions of ecteinascidin 743–DNA adducts by the UvrABC nuclease and the unique structural feature of the DNA adducts can be used to explain the repair-dependent toxicities of this antitumor agent

Zewail-Foote

Kohn

et al. 2001

Chemistry & Biology

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The inefficient repair incision by the UvrABC nuclease of Et 743-DNA adducts provides a basis for rationalizing the observed repair-dependent cytotoxicities of these DNA adducts, if other associated structural properties of Et 743-DNA adducts are taken into account. In particular, the wedge-shaped Et 743, which forces open the minor groove of DNA, introducing a major groove bend, and the extrahelical protrusion of the C-subunit of Et 743 provide unique characteristics alongside the hydrogen-bonding stabilization of a covalent DNA adduct, which we propose traps an intermediate in NER processing of Et 743-DNA adducts. This trapped intermediate protein-Et 743-DNA adduct complex can be considered analogous to a poisoned topoisomerase I- or topoisomerase II-DNA complex. In the absence of an intact NER nuclease complex, this toxic lesion is unable to form, and the Et 743-DNA adducts, although not repaired by the NER pathway, are less toxic to cells. Conversely, elevated levels of either of these nucleases should lead to enhanced Et 743 toxicity.

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“…D N A m ay be d am ag ed by a variety o f agents (R eard o n & S an ca r 1991). A fairly com m on feature at the site of dam ag e m ay be axial kinking, as is found in the case of a cisplatin ad d u c t, for exam ple (Visse et al 1991). T his could g enerate a feature th at is recognizable by certain proteins.…”

Section: Recognition Of Branched Dna Structures By Proteinsmentioning

confidence: 92%

Structure of the four-way DNA junction and its interaction with proteins

Duckett

Murchie

Giraud-Panis

et al. 1995

Phil. Trans. R. Soc. Lond. B

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The four-way DNA junction is an important intermediate in recombination processes; it is, the substrate for different enzyme activities. In solution, the junction adopts a right-handed, antiparallel-stacked X-structure formed by the pairwise coaxial-stacking of helical arms. The stereochemistry is determined by the juxtaposition of grooves and backbones, which is optimal when the smaller included angle is 60 degrees. The antiparallel structure has two distinct sides with major and minor groove-characteristics, respectively. The folding process requires the binding of metal cations, in the absence of which, the junction remains extended without helix-helix stacking. The geometry of the junction can be perturbed by the presence of certain base-base mispairs or phosphodiester discontinuities located at the point of strand exchange. The four-way DNA junction is selectively cleaved by a number of resolving enzymes. In a number of cases, these appear to recognize the minor groove face of the junction and are functionally divisible into activities that recognize and bind the junction, and a catalytic activity. Some possible mechanisms for the recognition of branched DNA structure are discussed.

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Uvr excision repair protein complex of Escherichia coli binds to the convex side of a cisplatin-induced kink in the DNA.

Cited by 59 publications

References 28 publications

In vitro reconstitution of an efficient nucleotide excision repair system using mesophilic enzymes from Deinococcus radiodurans

In vitro reconstitution of an efficient nucleotide excision repair system using mesophilic enzymes from Deinococcus radiodurans

The inefficiency of incisions of ecteinascidin 743–DNA adducts by the UvrABC nuclease and the unique structural feature of the DNA adducts can be used to explain the repair-dependent toxicities of this antitumor agent

Structure of the four-way DNA junction and its interaction with proteins

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