Aberrant mobility phenomena of the DNA repair protein XPA

Lm. Iakoucheva et al., Aberrant mobility phenomena of the DNA repair protein XPA, PROTEIN SCI, 10(7), 2001, pp. 1353-1362
Citations number
Categorie Soggetti
Biochemistry & Biophysics
Journal title
ISSN journal
0961-8368 → ACNP
Year of publication
1353 - 1362
SICI code
The DNA repair protein XPA recognizes a wide variety of bulky lesions and i nteracts 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 tim e-resolved trypsin proteolysis and electrospray ionization interface couple d to a Fourier transform ion cyclotron resonance (ESI-FTICR) mass spectrome try (MS). MS data were consistent with the interpretation that xXPA contain s no post-translational modifications. Here we characterize the discrepancy between the calculated molecular weight (31 kDa) for xXPA and its apparent molecular weight on SDS-PAGE (multiple bands from similar to 40-45 kDa) an d gel filtration chromatography (similar to 92 kDa), as well as the consequ ences of DNA binding on its anomalous mobility. Iodoacetamide treatment of xXPA prior to SDS-PAGE yielded a single 42-kDa band, showing that covalent modification of Cys did not correct aberrant mobility. Determination of sul fhydryl content in xXPA with Ellman's reagent revealed that all nine Cys in active protein are reduced. Unexpectedly, structural constraints induced b y intramolecular glutaraldehyde crosslinks in xXPA produced a similar to 32 -kDa monomer in closer agreement with its calculated molecular weight. To i nvestigate whether binding to DNA alters xXPA's anomalous migration, we use d gel filtration chromatography. For the first time, we purified stable com plexes of xXPA and DNA +/- cisplatin +/- mismatches. xXPA showed at least 1 0-fold higher affinity for cisplatin DNA +/- mismatches compared to undamag ed DNA F mismatches. In all cases, DNA binding did not correct xXPA's anoma lous migration. To test predictions that a Glu-rich region (EEEEAEE) and/or disordered N- and C-terminal domains were responsible for xXPA's aberrant mobility, the molecular weights of partial proteolytic fragments from simil ar to5 to 25 kDa separated by reverse-phase HPLC and precisely determined b y ESI-FTICR MS were correlated with their migration on SDS-PAGE, Every part ial tryptic fragment analyzed within this size range exhibited 10%-50% larg er molecular weights than expected, Thus, both the disordered domains and t he Glu-rich region in xXPA are primarily responsible for the aberrant mobil ity phenomena.