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Twist-open mechanism of DNA damage recognition by the Rad4/XPC nucleotide excision repair complex.

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journal contribution
posted on 01.06.2016, 00:00 by Y Velmurugu, X Chen, P Slogoff Sevilla
DNA damage repair starts with the recognition of damaged sites from predominantly normal DNA. In eukaryotes, diverse DNA lesions from environmental sources are recognized by the xeroderma pigmentosum C (XPC) nucleotide excision repair complex. Studies of Rad4 (radiation-sensitive 4; yeast XPC ortholog) showed that Rad4 "opens" up damaged DNA by inserting a β-hairpin into the duplex and flipping out two damage-containing nucleotide pairs. However, this DNA lesion "opening" is slow (˜5-10 ms) compared with typical submillisecond residence times per base pair site reported for various DNA-binding proteins during 1D diffusion on DNA. To address the mystery as to how Rad4 pauses to recognize lesions during diffusional search, we examine conformational dynamics along the lesion recognition trajectory using temperature-jump spectroscopy. Besides identifying the ˜10-ms step as the rate-limiting bottleneck towards opening specific DNA site, we uncover an earlier ˜100- to 500-μs step that we assign to nonspecific deformation (unwinding/"twisting") of DNA by Rad4. The β-hairpin is not required to unwind or to overcome the bottleneck but is essential for full nucleotide-flipping. We propose that Rad4 recognizes lesions in a step-wise "twist-open" mechanism, in which preliminary twisting represents Rad4 interconverting between search and interrogation modes. Through such conformational switches compatible with rapid diffusion on DNA, Rad4 may stall preferentially at a lesion site, offering time to open DNA. This study represents the first direct observation, to our knowledge, of dynamical DNA distortions during search/interrogation beyond base pair breathing. Submillisecond interrogation with preferential stalling at cognate sites may be common to various DNA-binding proteins.

Funding

This work was funded by NSF Grants MCB-0721937 and MCB-1158217 (to A.A.) and MCB-1412692 (to J.-H.M.), the Chancellor’s Discovery Fund (to A.A. and J.-H.M.), and a startup fund from the University of Illinois at Chicago (to J.-H.M.).

History

Publisher Statement

This is a copy of an article published in the Proceedings of the National Academy of Sciences © 2016 National Academy of Sciences Publications.

Publisher

National Academy of Sciences

Issue date

19/04/2016