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

Abstract

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...

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

“…Binding of UvrA to TRCF could not be detected by gel filtration (4), although an interaction could be detected by pull-down assays, which more readily capture transient interactions (9). In contrast, UvrB binding to UvrA is clearly detected by gel filtration (20). These previous results are in agreement with the small but conserved TRCF-UvrA interface that we observe.…”

“…UvrA residues interacting with TRCF include conserved E219, which is important for NER (20), as well as invariant R176 and R206, which also map to the interface and are critical for UvrA-UvrB complex formation (Fig. 2B) (20). The corollary of our crystallographic study is that, for UvrA to bind, the D2-D7 contacts must be broken during the coupling process either concomitant with or subsequent to TEC binding.…”

“…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).…”

“…Other conserved solvent-exposed residues in TRCF-Trunc contribute to the intramolecular contacts seen in full-length TRCF. UvrA residues interacting with TRCF include conserved E219, which is important for NER (20), as well as invariant R176 and R206, which also map to the interface and are critical for UvrA-UvrB complex formation (Fig. 2B) (20).…”

Surviving the Sun: Repair and bypass of DNA UV lesions

“…63 On the other hand, XPC clearly recognizes and binds the undamaged strand opposite a lesion. 35,61 DNA at the lesion site is severely distorted in all cases.…”