Crystal Structure of Bacillus stearothermophilus UvrA Provides Insight into ATP-Modulated Dimerization, UvrB Interaction, and DNA Binding

Pakotiprapha, Danaya; Inuzuka, Yoshihiko; Bowman, Brian R.; Moolenaar, Geri F.; Goosen, Nora; Jeruzalmi, David; Verdine, Gregory L.

doi:10.1016/j.molcel.2007.10.026

Cited by 80 publications

(149 citation statements)

References 68 publications

(74 reference statements)

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“…The resulting Mt UvrA•UvrB complex would be compatible with both an UvrA 2 •UvrB 2 (Figure 7b, upper panels) and an UvrA 2 •UvrB (not shown) stoichiometry. Moreover, the different conformation adopted by the UvrB-BD, respectively, in the structure of ligand-free Mt UvrA, Bst UvrA•ADP (16) and Tm UvrA•DNA (17) (Figure 7b, from top to bottom), appears in all cases competent for the concomitant association to UvrB and DNA, although with different geometry. In fact, while in the Mt UvrA 2 •UvrB 2 model the double-strand DNA should be hosted inside an only partially solvent-accessible track, which is built up by the facing ventral surfaces of both protein components, in the other two models the UvrB-BD repositioning would result in the exposure of the DNA molecule to the bulk solvent.…”

Section: Resultsmentioning

confidence: 97%

The biological and structural characterization of Mycobacterium tuberculosis UvrA provides novel insights into its mechanism of action

Rossi

Khanduja

Bortoluzzi

et al. 2011

Nucleic Acids Research

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Mycobacterium tuberculosis is an extremely well adapted intracellular human pathogen that is exposed to multiple DNA damaging chemical assaults originating from the host defence mechanisms. As a consequence, this bacterium is thought to possess highly efficient DNA repair machineries, the nucleotide excision repair (NER) system amongst these. Although NER is of central importance to DNA repair in M. tuberculosis, our understanding of the processes in this species is limited. The conserved UvrABC endonuclease represents the multi-enzymatic core in bacterial NER, where the UvrA ATPase provides the DNA lesion-sensing function. The herein reported genetic analysis demonstrates that M. tuberculosis UvrA is important for the repair of nitrosative and oxidative DNA damage. Moreover, our biochemical and structural characterization of recombinant M. tuberculosis UvrA contributes new insights into its mechanism of action. In particular, the structural investigation reveals an unprecedented conformation of the UvrB-binding domain that we propose to be of functional relevance. Taken together, our data suggest UvrA as a potential target for the development of novel anti-tubercular agents and provide a biochemical framework for the identification of small-molecule inhibitors interfering with the NER activity in M. tuberculosis.

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Section: Resultsmentioning

confidence: 97%

The biological and structural characterization of Mycobacterium tuberculosis UvrA provides novel insights into its mechanism of action

Rossi

Khanduja

Bortoluzzi

et al. 2011

Nucleic Acids Research

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“…Recent studies suggest that domain D2 of TRCF interacts with an UvrA fragment encompassing residues 131-250 (9,18,20,22,25). Furthermore, the UvrB-homology module (residues 1-349, including D2) as well as residues 131-248 of UvrA were shown to be essential for repair of the template strand in vitro and in vivo without being required for RNAP displacement (21).…”

Section: Resultsmentioning

confidence: 99%

Nucleotide excision repair (NER) machinery recruitment by the transcription-repair coupling factor involves unmasking of a conserved intramolecular interface

Deaconescu

Sevostyanova²,

Artsimovitch³

et al. 2012

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

100

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Transcription-coupled DNA repair targets DNA lesions that block progression of elongating RNA polymerases. In bacteria, the transcription-repair coupling factor (TRCF; also known as Mfd) SF2 ATPase recognizes RNA polymerase stalled at a site of DNA damage, removes the enzyme from the DNA, and recruits the Uvr(A)BC nucleotide excision repair machinery via UvrA binding. Previous studies of TRCF revealed a molecular architecture incompatible with UvrA binding, leaving its recruitment mechanism unclear. Here, we examine the UvrA recognition determinants of TRCF using X-ray crystallography of a core TRCF-UvrA complex and probe the conformational flexibility of TRCF in the absence and presence of nucleotides using small-angle X-ray scattering. We demonstrate that the C-terminal domain of TRCF is inhibitory for UvrA binding, but not RNA polymerase release, and show that nucleotide binding induces concerted multidomain motions. Our studies suggest that autoinhibition of UvrA binding in TRCF may be relieved only upon engaging the DNA damage.cysteine cross-linking | transcription | ATPase stimulation | UvrB R NA polymerase (RNAP) stalled at DNA lesions on the transcribed strand elicits a preferential pathway for nucleotide excision repair (NER) called transcription-coupled repair (TCR), which is present in Bacteria and Eukarya (1). Bacterial transcription-repair coupling factor (TRCF; also known as Mfd) orchestrates this process by specific recognition of the transcription and NER assemblies, which reflects its twofold role. First, TRCF relieves transcription-dependent NER inhibition due to occlusion of the DNA lesion by RNAP (2). TRCF, an SF2 ATPase with dsDNA translocase but no helicase activity (3), approaches the stalled RNAP from behind and induces its forward translocation by stepping on dsDNA using ATP hydrolysis (4, 5). The consequent collapse of the upstream end of the transcription bubble leads to massive destabilization of the otherwise stable ternary elongation complex (TEC) and transcription termination (4-7). Rho, the only other known bacterial enzymatic terminator, induces termination by a similar forward-translocation mechanism, but translocates along the nascent RNA (8). Second, TRCF recruits the Uvr(A)BC endonuclease to the unmasked lesion by binding to UvrA (4, 9). This initiates a cascade of events resulting in lesion excision and gap filling (4, 10). TRCF also has roles beyond TCR-in the rescue of replication forks stalled by head-on collisions with RNAPs (11), in the development of antibiotic resistance (12, 13), recombination (14, 15), and transcriptional regulation (16, 17).The crystal structure of apo TRCF (18) revealed a multimodular enzyme with eight domains connected by flexible linkers (Fig. 1A), an architecture that appears primed for large conformational changes, which are believed to be critical for coupling RNAP recognition to recruitment of NER enzymes. Domains D1 and D2 of TRCF are similar to the NER protein UvrB, which also binds UvrA (18,19), suggesting that these domains serve as a pl...

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“…Recently, we reported the structure of Geobacillus stearothermophilus UvrA and the identification of binding sites for DNA and UvrB (5). We also established that the identified UvrB-binding domain is necessary and sufficient to mediate the UvrA-UvrB interaction and that the isolated interaction domains of UvrA (5) and UvrB (6) bind to each other in solution.…”

mentioning

confidence: 83%

A Structural Model for the Damage-sensing Complex in Bacterial Nucleotide Excision Repair

Pakotiprapha

Liu

Verdine

et al. 2009

Journal of Biological Chemistry

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Nucleotide excision repair is distinguished from other DNA repair pathways by its ability to process a wide range of structurally unrelated DNA lesions. In bacteria, damage recognition is achieved by the UvrA⅐UvrB ensemble. Here, we report the structure of the complex between the interaction domains of UvrA and UvrB. These domains are necessary and sufficient for fulllength UvrA and UvrB to associate and thereby form the DNA damage-sensing complex of bacterial nucleotide excision repair. The crystal structure and accompanying biochemical analyses suggest a model for the complete damage-sensing complex.Nucleotide excision repair is distinguished from other DNA repair pathways by its ability to process a diverse set of lesions. In bacteria, the initial steps are carried out by three proteins: UvrA, UvrB, and UvrC. The UvrA⅐UvrB complex conducts surveillance of DNA and recognizes damage. Having located a lesion, UvrA "loads" UvrB onto the DNA at the damaged sites and then dissociates. Damage searching, formation of the UvrB⅐DNA "preincision" complex, and dissociation of UvrA are regulated by ATP (1). UvrB subsequently recruits the endonuclease UvrC, which catalyzes incisions on either side of the lesion (2, 3). Following incision, UvrC and the damage-containing oligonucleotide are removed by UvrD (helicase II), whereas UvrB remains bound to the gapped DNA and recruits DNA polymerase I for repair synthesis. Sealing of the single-stranded nick completes the repair process and restores the original DNA sequence (4).Since its discovery more than 40 years ago, bacterial nucleotide excision repair has been extensively studied, resulting in a large body of work that describes the protein components and the details of how they operate. Notwithstanding the trove of genetic and biochemical data, several key questions remain unanswered. For example, how does the same set of proteins handle a diverse set of lesions while maintaining specificity? How do UvrA and UvrB cooperate during damage recognition, and what is the precise role of ATP? Ongoing studies in the field, including those described below, aim to address these issues.Recently, we reported the structure of Geobacillus stearothermophilus UvrA and the identification of binding sites for DNA and UvrB (5). We also established that the identified UvrB-binding domain is necessary and sufficient to mediate the UvrA-UvrB interaction and that the isolated interaction domains of UvrA (5) and UvrB (6) bind to each other in solution.To understand the interaction between UvrA and UvrB, we have determined the crystal structure of the complex between the two isolated interaction domains. The structure revealed that UvrA-UvrB interaction interface is largely polar, mediated by several highly conserved charged residues. Site-directed mutagenesis and biochemical characterization of the mutant proteins confirmed the importance of the observed interactions. Based on the interaction domain complex structure, we have constructed a structural model for the full-length UvrA⅐UvrB ensemble an...

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Crystal Structure of Bacillus stearothermophilus UvrA Provides Insight into ATP-Modulated Dimerization, UvrB Interaction, and DNA Binding

Cited by 80 publications

References 68 publications

The biological and structural characterization of Mycobacterium tuberculosis UvrA provides novel insights into its mechanism of action

The biological and structural characterization of Mycobacterium tuberculosis UvrA provides novel insights into its mechanism of action

Nucleotide excision repair (NER) machinery recruitment by the transcription-repair coupling factor involves unmasking of a conserved intramolecular interface

A Structural Model for the Damage-sensing Complex in Bacterial Nucleotide Excision Repair

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