DNA wrapping is required for DNA damage recognition in the Escherichia coli DNA nucleotide excision repair pathway

Abstract

Localized DNA melting may provide a general strategy for recognition of the wide array of chemically and structurally diverse DNA lesions repaired by the nucleotide excision repair (NER) pathway. However, it is not clear what causes such DNA melting and how it is driven. Here, we show a DNA wrapping-melting model supported by results from dynamic monitoring of the key DNA-protein and protein-protein interactions involved in the early stages of the Escherichia coli NER process. Using an analytical technique involving capillary electrophoresis coupled with laser-induced fluorescence polarization, which combines a mobility shift assay with conformational analysis, we demonstrate that DNA wrapping around UvrB, mediated by UvrA, is an early event in the damage-recognition process during E. coli NER. DNA wrapping of UvrB was confirmed by Fö rster resonance energy transfer and fluorescence lifetime measurements. This wrapping did not occur with readily denaturable damaged DNA substrates (''bubble'' DNA), suggesting that DNA wrapping of UvrB plays an important role in the induction of DNA melting around the damage site. Analysis of DNA wrapping of mutant UvrB Y96A further suggests that a cooperative interaction between DNA wrapping of UvrA2B and contact of the ␤-hairpin of UvrB with the bulky damage moiety may be involved in the local DNA melting at the damage site.capillary electrophoresis ͉ laser induced fluorescence polarization ͉ Bacillus caldotenax ͉ fluorescence resonance energy transfer N ucleotide excision repair (NER) displays a unique capability of recognizing and processing a broad spectrum of bulky lesions, which are chemically and structurally diverse (1, 2). In Escherichia coli, the complex of UvrA and UvrB is responsible for damage search and recognition along DNA (1, 3). A number of models have been proposed to explain the repair of the broad spectrum of substrates acted on by the E. coli NER, including the helicasescanning model (4-6), the damage-processing model (7), and the padlock model (8, 9). The key difference among these models is the mechanism of damage recognition. In the helicase-scanning model, the UvrA 2 B complex locates damage through a helicase-driven translocation step. In the damage-processing model, the UvrA 2 B complex finds damage by random diffusion. Accumulating evidence shows that local DNA melting around the damage site induced by the UvrAB complex provides a structural basis for efficient incision of damaged nucleotides by the UvrB and UvrC proteins (10-12). These observations form the basis for the padlock model. Recently, crystal structure analysis of UvrB-DNA complexes has provided evidence that also supports the padlock model (13,14). However, the mechanism responsible for DNA melting remains to be elucidated. It has been proposed that DNA wrapping around NER proteins may be pertinent to the recognition of the broad spectrum of repairable substrates (15-17). There is a need to confirm this proposition with experiments conducted under dynamic conditions.In the present study, ...

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