西亚试剂：DNA wrapping is required for DNA damage recognition in the

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

Hailin Wanga,b, Meiling Lua, Moon-shong Tangc, Bennett Van Houtend,1, J. B. Alexander Rosse, Michael Weinfeldf,2 and X. Chris Lea,2

aDepartment of Laboratory Medicine and Pathology, University of Alberta, Edmonton, AB, Canada T6G 2G3;
bState Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China;
cDepartment of Environmental Medicine, New York University, Tuxedo, NY 10987;
dNational Institute of Environmental Health Sciences, Research Triangle Park, NC 27709;
eDepartment of Chemistry and Biochemistry and BioSpectroscopy Core Research Laboratory, University of Montana, Missoula, MT 59812; and
fExperimental Oncology, Cross Cancer Institute, Edmonton, AB, Canada T6G 1Z2

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 F?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 β-hairpin of UvrB with the bulky damage moiety may be involved in the local DNA melting at the damage site.

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