Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
Protein Sci
2001 Jul 01;107:1353-62. doi: 10.1110/ps.ps.40101.
Show Gene links
Show Anatomy links
Aberrant mobility phenomena of the DNA repair protein XPA.
Iakoucheva LM
,
Kimzey AL
,
Masselon CD
,
Smith RD
,
Dunker AK
,
Ackerman EJ
.
???displayArticle.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 approximately 40-45 kDa) and gel filtration chromatography ( approximately 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 approximately 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 approximately 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.
Ackerman,
Nucleotide excision repair in oocyte nuclear extracts from Xenopus laevis.
2000, Pubmed,
Xenbase
Ackerman,
Nucleotide excision repair in oocyte nuclear extracts from Xenopus laevis.
2000,
Pubmed
,
Xenbase
Armstrong,
The anomalous electrophoretic behavior of the human papillomavirus type 16 E7 protein is due to the high content of acidic amino acid residues.
1993,
Pubmed
Asahina,
The XPA protein is a zinc metalloprotein with an ability to recognize various kinds of DNA damage.
1994,
Pubmed
Billings,
Anomalous electrophoretic mobility of Drosophila phosphorylated H1 histone: is it related to the compaction of satellite DNA into heterochromatin?
1979,
Pubmed
Bordini,
Probing the reactivity of S-S bridges to acrylamide in some proteins under high pH conditions by matrix-assisted laser desorption/ ionisation.
1999,
Pubmed
Buchko,
Structural features of the minimal DNA binding domain (M98-F219) of human nucleotide excision repair protein XPA.
1998,
Pubmed
Buchko,
Extended X-ray absorption fine structure evidence for a single metal binding domain in Xenopus laevis nucleotide excision repair protein XPA.
1999,
Pubmed
,
Xenbase
Buschta-Hedayat,
Recognition of nonhybridizing base pairs during nucleotide excision repair of DNA.
1999,
Pubmed
Dunker,
Observations on molecular weight determinations on polyacrylamide gel.
1969,
Pubmed
Dunker,
Mobility of sodium dodecyl sulphate - protein complexes.
1976,
Pubmed
Fontana,
Probing the conformational state of apomyoglobin by limited proteolysis.
1997,
Pubmed
Hess,
Bipartite substrate discrimination by human nucleotide excision repair.
1997,
Pubmed
Hu,
The conserved, hydrophilic and arginine-rich N-terminal domain of cucumovirus coat proteins contributes to their anomalous electrophoretic mobilities in sodium dodecylsulfate-polyacrylamide gels.
1995,
Pubmed
Iakoucheva,
Identification of intrinsic order and disorder in the DNA repair protein XPA.
2001,
Pubmed
,
Xenbase
Ikegami,
Solution structure of the DNA- and RPA-binding domain of the human repair factor XPA.
1998,
Pubmed
Jones,
Preferential binding of the xeroderma pigmentosum group A complementing protein to damaged DNA.
1993,
Pubmed
Klenova,
Molecular weight abnormalities of the CTCF transcription factor: CTCF migrates aberrantly in SDS-PAGE and the size of the expressed protein is affected by the UTRs and sequences within the coding region of the CTCF gene.
1997,
Pubmed
Kriwacki,
Structural studies of p21Waf1/Cip1/Sdi1 in the free and Cdk2-bound state: conformational disorder mediates binding diversity.
1996,
Pubmed
Kriwacki,
Probing protein structure using biochemical and biophysical methods. Proteolysis, matrix-assisted laser desorption/ionization mass spectrometry, high-performance liquid chromatography and size-exclusion chromatography of p21Waf1/Cip1/Sdi1.
1997,
Pubmed
Kuraoka,
Identification of a damaged-DNA binding domain of the XPA protein.
1996,
Pubmed
Laemmli,
Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
1970,
Pubmed
Lao,
Replication protein A interactions with DNA. III. Molecular basis of recognition of damaged DNA.
2000,
Pubmed
Lindahl,
Quality control by DNA repair.
1999,
Pubmed
Mach,
Statistical determination of the average values of the extinction coefficients of tryptophan and tyrosine in native proteins.
1992,
Pubmed
Marciani,
Anomalous behavior of the major avian myeloblastosis virus glycoprotein in the presence of sodium dodecyl sulfate.
1978,
Pubmed
Miyamoto,
Mutational analysis of the structure and function of the xeroderma pigmentosum group A complementing protein. Identification of essential domains for nuclear localization and DNA excision repair.
1992,
Pubmed
Moggs,
Differential human nucleotide excision repair of paired and mispaired cisplatin-DNA adducts.
1997,
Pubmed
Mu,
Recognition and repair of compound DNA lesions (base damage and mismatch) by human mismatch repair and excision repair systems.
1997,
Pubmed
Nettleton,
Probing conformations of amyloidogenic proteins by hydrogen exchange and mass spectrometry.
1999,
Pubmed
Oda,
DNA polymerases alpha and beta are required for DNA repair in an efficient nuclear extract from Xenopus oocytes.
1996,
Pubmed
,
Xenbase
Pitt-Rivers,
The binding of sodium dodecyl sulphate to various proteins.
1968,
Pubmed
Poduslo,
Molecular weight estimation using sodium dodecyl sulfate--pore gradient electrophoresis.
1980,
Pubmed
Query,
A common RNA recognition motif identified within a defined U1 RNA binding domain of the 70K U1 snRNP protein.
1989,
Pubmed
Redeker,
Probing the native structure of stathmin and its interaction domains with tubulin. Combined use of limited proteolysis, size exclusion chromatography, and mass spectrometry.
2000,
Pubmed
Reynolds,
The gross conformation of protein-sodium dodecyl sulfate complexes.
1970,
Pubmed
Samsó,
Evidence for sodium dodecyl sulfate/protein complexes adopting a necklace structure.
1995,
Pubmed
Shirahama,
Free-boundary electrophoresis of sodium dodecyl sulfate-protein polypeptide complexes with special reference to SDS-polyacrylamide gel electrophoresis.
1974,
Pubmed
States,
Distribution of mutations in the human xeroderma pigmentosum group A gene and their relationships to the functional regions of the DNA damage recognition protein.
1998,
Pubmed
Westerhuis,
Reevaluation of the electrophoretic migration behavior of soluble globular proteins in the native and detergent-denatured states in polyacrylamide gels.
2000,
Pubmed
Wright,
Intrinsically unstructured proteins: re-assessing the protein structure-function paradigm.
1999,
Pubmed
Xu,
Electrospray ionization-mass spectrometry study of the interaction of cisplatin-adducted oligonucleotides with human XPA minimal binding domain protein.
1999,
Pubmed
