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Recognition of helical kinks by xeroderma pigmentosum group A protein triggers DNA excision repair

Nature Structural & Molecular Biology volume 13, pages 278–284 (2006)Cite this article

Abstract

The function of human XPA protein, a key subunit of the nucleotide excision repair pathway, has been examined with site-directed substitutions in its putative DNA-binding cleft. After screening for repair activity in a host-cell reactivation assay, we analyzed mutants by comparing their affinities for different substrate architectures, including DNA junctions that provide a surrogate for distorted reaction intermediates, and by testing their ability to recruit the downstream endonuclease partner. Normal repair proficiency was retained when XPA mutations abolished only the simple interaction with linear DNA molecules. By contrast, results from a K141E K179E double mutant revealed that excision is crucially dependent on the assembly of XPA protein with a sharp bending angle in the DNA substrate. These findings show how an increased deformability of damaged sites, leading to helical kinks recognized by XPA, contributes to target selectivity in DNA repair.

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Figure 1: Structure of human XPA protein.
Figure 2: Screening for DNA repair activity by host-cell reactivation assay.
Figure 3: DNA-binding properties of XPA mutants.
Figure 4: DNA-binding profiles of XPA tandem mutants.
Figure 5: Photo–cross-linking to the kinked junction region.
Figure 6: Recruitment of XPF–ERCC1 and dimerization.

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Acknowledgements

We thank M. Vitanescu for excellent technical assistance, J. Kaspàrkovà (Academy of Sciences of the Czech Republic) for the gift of cisplatin-modified oligonucleotides and O. Schärer (State University of New York, Stony Brook) for the gift of XPF–ERCC1. This work was supported by Swiss National Science Foundation grant 3100A-101747.

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Authors and Affiliations

  1. Institute of Pharmacology and Toxicology, University of Zürich-Vetsuisse, Winterthurerstrasse 260, Zürich, CH-8057, Switzerland

    Ulrike Camenisch, Ramiro Dip, Sylvie Briand Schumacher & Hanspeter Naegeli

  2. Institute of Biochemistry, University of Zürich, Winterthurerstrasse 190, Zürich, CH-8057, Switzerland

    Benjamin Schuler

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  1. Ulrike Camenisch
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Correspondence to Hanspeter Naegeli.

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Supplementary information

Supplementary Fig. 1

Screening of XPA single mutants (PDF 84 kb)

Supplementary Fig. 2

Purity of XPA proteins (PDF 86 kb)

Supplementary Fig. 3

Sequence alignment of XPA homologs (PDF 45 kb)

Supplementary Table 1

Oligonucleotide primers for mutagenesis (PDF 56 kb)

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Camenisch, U., Dip, R., Schumacher, S. et al. Recognition of helical kinks by xeroderma pigmentosum group A protein triggers DNA excision repair. Nat Struct Mol Biol 13, 278–284 (2006). https://doi.org/10.1038/nsmb1061

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