Aberrant mobility phenomena of the DNA repair protein XPA

Lilia M. Iakoucheva, Amy L. Kimzey, Christophe D. Masselon, Richard D. Smith, A. Dunker, Eric J. Ackerman

Research output: Contribution to journalArticle

91 Citations (Scopus)

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). MS data were consistent with the interpretation that xXPA contains 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 ∼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, showing 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 intramolecular glutaraldehyde crosslinks in xXPA produced a ∼32-kDa monomer in closer agreement with its calculated molecular weight. 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. 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 ∼5 to 25 kDa separated by reverse-phase HPLC and precisely determined by ESI-FTICR MS were correlated with their migration on SDS-PAGE. Every partial tryptic fragment analyzed within this size range exhibited 10%-50% larger molecular weights than expected. Thus, both the disordered domains and the Glu-rich region in xXPA are primarily responsible for the aberrant mobility phenomena.

Original languageEnglish (US)
Pages (from-to)1353-1362
Number of pages10
JournalProtein Science
Volume10
Issue number7
DOIs
StatePublished - 2001
Externally publishedYes

Fingerprint

Xenopus
DNA Repair
Repair
DNA
Molecular weight
Molecular Weight
Proteins
Mass spectrometry
Chromatography
Polyacrylamide Gel Electrophoresis
Cisplatin
Electrospray Ionization Mass Spectrometry
Gels
Xenopus Proteins
Proteolysis
Gel Chromatography
Dithionitrobenzoic Acid
Iodoacetamide
Cyclotron resonance
Electrospray ionization

Keywords

  • DNA repair
  • ESI-FTICR mass spectrometry
  • Gel electrophoresis
  • Intrinsic disorder
  • Partial proteolysis
  • SDS-PAGE
  • XPA

ASJC Scopus subject areas

  • Biochemistry

Cite this

Iakoucheva, L. M., Kimzey, A. L., Masselon, C. D., Smith, R. D., Dunker, A., & Ackerman, E. J. (2001). Aberrant mobility phenomena of the DNA repair protein XPA. Protein Science, 10(7), 1353-1362. https://doi.org/10.1110/ps.40101

Aberrant mobility phenomena of the DNA repair protein XPA. / Iakoucheva, Lilia M.; Kimzey, Amy L.; Masselon, Christophe D.; Smith, Richard D.; Dunker, A.; Ackerman, Eric J.

In: Protein Science, Vol. 10, No. 7, 2001, p. 1353-1362.

Research output: Contribution to journalArticle

Iakoucheva, LM, Kimzey, AL, Masselon, CD, Smith, RD, Dunker, A & Ackerman, EJ 2001, 'Aberrant mobility phenomena of the DNA repair protein XPA', Protein Science, vol. 10, no. 7, pp. 1353-1362. https://doi.org/10.1110/ps.40101
Iakoucheva LM, Kimzey AL, Masselon CD, Smith RD, Dunker A, Ackerman EJ. Aberrant mobility phenomena of the DNA repair protein XPA. Protein Science. 2001;10(7):1353-1362. https://doi.org/10.1110/ps.40101
Iakoucheva, Lilia M. ; Kimzey, Amy L. ; Masselon, Christophe D. ; Smith, Richard D. ; Dunker, A. ; Ackerman, Eric J. / Aberrant mobility phenomena of the DNA repair protein XPA. In: Protein Science. 2001 ; Vol. 10, No. 7. pp. 1353-1362.
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