Analysis of UvrABC endonuclease reaction intermediates on cisplatin-damaged DNA using mobility shift gel electrophoresis.

Visse, Robert; Ruijter, M de; Moolenaar, Geri F.; Putte, Pieter van de

doi:10.1016/s0021-9258(19)50487-2

Cited by 77 publications

(44 citation statements)

References 23 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: 67%

“…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: 67%

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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“…Binding of UvrC to the preincision complex is essential for incision to take place. Although it is generally believed that UvrC binds to the complex after the release of UvrA, we have shown that preincision complexes still containing UvrA are also incisionproficient (Visse et al, 1992). The DNA is incised asymmetrically on both sides of the lesion, first the 3' incision 4-5 t This work was supported by the J.…”

mentioning

confidence: 59%

“…This complex is extremely stable with a half-life of 55 min (Yeung et al, 1986). It was found that the preincision complex contains only UvrB (Orren & Sancar, 1989,1990Visse et al, 1992) which was surprising as UvrB alone has no affinity for (damaged) DNA. Apparently conformational changes in the UvrA2B-DNA complex allow UvrB binding.…”

mentioning

confidence: 99%

The Actual Incision Determines the Efficiency of Repair of Cisplatin-Damaged DNA by the Escherichia coli UvrABC Endonuclease

Visse¹,

Gool²,

Moolenaar³

et al. 1994

Biochemistry

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The UvrABC endonuclease from Escherichia coli repairs a broad spectrum of DNA lesions with variable efficiencies. The effectiveness of repair is influenced by the nature of the lesion, the local DNA sequence, and/or the topology of the DNA. To get a better understanding of the aspects of this multistep repair reaction that determine the effectiveness of repair, we compared the incision efficiencies of linear DNA fragments containing either a site-specific cis-[Pt(NH3)2(d(GpG)-N7(1),-N7(2)]] or a cis- Pt(NH3)2[d(GpCpG)-N7(1),-N7(3)]] adduct. Overall the DNA with the cis-PtGG adduct was incised about 3.5 times more efficiently than the cis-Pt.GCG-containing DNA. The rate of UvrB-DNA preincision complex formation for both lesions was similar and high in relation to the incision. DNase I footprints, however, showed that the local structure of the two preincision complexes is different. An assay was developed to measure the binding of UvrC to the preincision complexes and it was found that the binding rate of UvrC to the more slowly incised cis-Pt.GCG preincision complex was higher than to the cis-Pt.GG preincision complex. This most likely reflects a qualitative difference in preincision complex structures. For both lesions the binding of UvrC to the preincision complex was fast compared to the kinetics of actual incision. Apparently, direct incision of cisplatin damage requires an additional conformational change after the binding of UvrC.

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“…To continue characterizing UvrC in holo form, we examined how UvrC interacts with radiolabeled, duplexed 30 base pair substrates using electrophoretic mobility shift assays (EMSAs) completed in an anaerobic chamber. ,,, UvrC along with the full UvrABC exinuclease has been studied extensively in vitro with single-stranded DNA (ssDNA), well-matched (WM) double-stranded DNA (dsDNA), damaged duplex substrates, and substrates with nicks, gaps, bubbled regions, and overhangs derived from plasmid DNA and synthetic oligomers. , The majority of previous work from chromatographic, optical, and gel-based methods with UvrC from E. coli and thermophilic bacteria indicates that UvrC does not form a complex with dsDNA independently of UvrA and UvrB. − (It should be noted that UvrC, both truncated and full-length, has been seen to bind to ssDNA or single-stranded regions of nicked, gapped, or bubbled substrates. ,,− ) Thus, it is widely accepted that UvrC requires the action(s) of UvrA and UvrB in order to associate with substrates of duplex character.…”

Section: Resultsmentioning

confidence: 99%

UvrC Coordinates an O₂-Sensitive [4Fe4S] Cofactor

2020

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Recent advances have led to numerous landmark discoveries of [4Fe4S] clusters coordinated by essential enzymes in repair, replication, and transcription across all domains of life. The cofactor has notably been challenging to observe for many nucleic acid processing enzymes due to several factors, including a weak bioinformatic signature of the coordinating cysteines and lability of the metal cofactor. To overcome these challenges, we have used sequence alignments, an anaerobic purification method, iron quantification, and UV–visible and electron paramagnetic resonance spectroscopies to investigate UvrC, the dual-incision endonuclease in the bacterial nucleotide excision repair (NER) pathway. The characteristics of UvrC are consistent with [4Fe4S] coordination with 60–70% cofactor incorporation, and additionally, we show that, bound to UvrC, the [4Fe4S] cofactor is susceptible to oxidative degradation with aggregation of apo species. Importantly, in its holo form with the cofactor bound, UvrC forms high affinity complexes with duplexed DNA substrates; the apparent dissociation constants to well-matched and damaged duplex substrates are 100 ± 20 nM and 80 ± 30 nM, respectively. This high affinity DNA binding contrasts reports made for isolated protein lacking the cofactor. Moreover, using DNA electrochemistry, we find that the cluster coordinated by UvrC is redox-active and participates in DNA-mediated charge transport chemistry with a DNA-bound midpoint potential of 90 mV vs NHE. This work highlights that the [4Fe4S] center is critical to UvrC.

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Analysis of UvrABC endonuclease reaction intermediates on cisplatin-damaged DNA using mobility shift gel electrophoresis.

Cited by 77 publications

References 23 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 Actual Incision Determines the Efficiency of Repair of Cisplatin-Damaged DNA by the Escherichia coli UvrABC Endonuclease

UvrC Coordinates an O₂-Sensitive [4Fe4S] Cofactor

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Analysis of UvrABC endonuclease reaction intermediates on cisplatin-damaged DNA using mobility shift gel electrophoresis.

Cited by 77 publications

References 23 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 Actual Incision Determines the Efficiency of Repair of Cisplatin-Damaged DNA by the Escherichia coli UvrABC Endonuclease

UvrC Coordinates an O2-Sensitive [4Fe4S] Cofactor

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UvrC Coordinates an O₂-Sensitive [4Fe4S] Cofactor