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

Abstract

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.

Alkaline “Nanoswords” Coordinate Ferroptosis-like Bacterial Death for Antibiosis and Osseointegration

“…DNA replication is essential and highly conserved, and the large number of proteins interact to replicate DNA in bacteria. If DNA is damaged, its timely repair is essential for microbe survival. − So, some DEGs associated with DNA synthesis, replication, and repair were further analyzed. The expression levels of IUJ41_08430 and Novel00040 are significantly downregulated for the MH group compared with the Ti group, indicating the restrained DNA synthesis, replication, and repair systems of S. aureus by MH compared with Ti (Figure g).…”

Structural and functional insights into the activation of the dual incision activity of UvrC, a key player in bacterial NER

“…Cloning, expression and purification of D. radiodurans UvrA1 (DR_1771), UvrB (DR_2275) and UvrC (DR_1354) have been described previously ( 45 ). Truncated constructs (Table S1) of UvrC (DR_1354) were cloned into pProexHtB (EMBL) for expression with a cleavable N-terminal His-tag.…”

“…All constructs were expressed in E. coli BL21 cells and expression was induced at 20°C overnight with 1 mM isopropyl- β-D-thiogalactopyranoside (IPTG). UvrC-Δ(HhH) 2 , UvrC E72A and UvrC D391A were purified as for UvrC wild-type ( 45 ). For the anaerobic purification of UvrC, all steps (from cell lysis to protein freezing at –80°C for long-term storage) were performed in a glovebox with oxygen levels below 5 ppm.…”

“…Incision activity measurements were performed using duplexed 50 mer dsDNA oligonucleotides composed of a 5′-ATTO633-labelled strand containing a fluorescein conjugated thymine in position 26 (strand name: 5′-ATTO633-F26-seq1) annealed with an unlabelled complementary strand (strand name: Rev-seq1). As described previously ( 45 ), for the incision assay, 25 nM F26-seq1 dsDNA substrate was incubated at 37°C for 5 min with 1 μM UvrA1, 0.5 μM UvrB and 2 μM UvrC in 50 mM Tris–HCl pH7.5, 50 mM KCl, 5 mM DTT and 2.5 mM MgCl 2 , before initiating the incision reaction with the addition of 2.5 mM ATP. The reactions were stopped by addition of 10 μl stop buffer to 10 μl reaction mix and subsequent heating of the samples to 95°C for 5 min.…”

“…Bacterial NER has been successfully reconstituted in vitro using the three essential UvrA, UvrB and UvrC proteins from either E. coli ( 39–41 ) or more recently from thermophilic bacteria ( B. caldotenax , Geobacillus stearothermophilus, T. maritima and T. thermophilus ) ( 4 , 15 , 25 , 26 , 34 , 42 , 43 ), and either lesion-containing plasmid or DNA oligonucleotides as substrates ( 39–42 , 44 ). We recently developed a highly efficient incision assay ( 45 ) relying on the activity of UvrA, UvrB and UvrC from a single organism, the mesophilic and radiation resistant bacterium, Deinococcus radiodurans that possesses a well-conserved NER system ( 46 ). In the present study, we have used this assay, together with biochemical, mutational, biophysical and structural studies, to (i) unveil the nature and particularities of the FeS cluster of D. radiodurans UvrC (DrUvrC), (ii) provide important insight into the regulation and coupling of the incision activities of DrUvrC, (iii) identify a remarkable feature of the N-terminal region of DrUvrC that is capable of catalyzing the dual incision reaction in the absence of the C-terminal endonuclease domain of UvrC, and (iv) establish the first complete three-dimensional model of a UvrC protein and propose a model of DNA-bound UvrC.…”