July 1, 2001
Journal Article

Aberrant mobility phenomena of the DNA repair protein XPA

Abstract

The DNA repair protein XPA recognizes a wide variety of bulky lesions and interacts with several other proteins during nucleotide excision repair. We recently identified regions of intrinsic order and disorder in full length Xenopus XPA (xXPA) protein using an experimental approach that combined time-resolved trypsin proteolysis and electrospray ionization interface coupled to a Fourier transform ion cyclotron resonance (ESI-FTICR) mass spectrometry (MS) (Iakoucheva, et al., Prot. Sci. 10: 560-571 (2001)). MS data were consistent with the interpretation that xXPA contains no post-translational modifications. Here we characterize the discrepancy between the calculated MW (31 kDa) for xXPA and its apparent MW on SDS-PAGE (multiple bands from ~40-45 kDa) and gel filtration chromatography (~92 kDa), as well as the consequences of DNA binding on its anomalous mobility. Iodoacetamide treatment of xXPA prior to SDS-PAGE yielded a single 42-kDa band, demonstrating that covalent modification of Cys did not correct aberrant mobility. Determination of sulfhydryl content in xXPA with Ellman's reagent revealed that all nine Cys in active protein are reduced. Unexpectedly, structural constraints induced by intra-molecular glutaraldehyde crosslinks in xXPA produced a ~32 kDa monomer in closer agreement with its calculated MW. To investigate whether binding to DNA alters xXPA's anomalous migration, we used gel filtration chromatography. For the first time, we purified stable complexes of xXPA and DNA ? cisplatin ? mismatches. xXPA showed at least 10-fold higher affinity for cisplatin DNA ? mismatches compared to undamaged DNA ? mismatches. In all cases, DNA binding did not correct xXPA's anomalous migration.

Revised: September 10, 2001 | Published: July 1, 2001

Citation

Iakoucheva L.M., A.L. Kimzey, C.D. Masselon, R.D. Smith, A.K. Dunker, and E.J. Ackerman. 2001. Aberrant mobility phenomena of the DNA repair protein XPA. Protein Science 10, no. 7:1353-1362. PNWD-SA-5300.