XPC and human homologs of RAD23: intracellular localization and relationship to other nucleotide excision repair complexes.
The xeroderma pigmentosum syndrome complementation group C (XP-C) is due to a defect in the global genome repair subpathway of nucleotide excision repair (NER). The XPC protein is complexed with HHR23B, one of the two human homologs of the yeast NER protein, RAD23 (Masutani at al. (1994) EMBO J. 8, 1831-1843). Using heparin chromatography, gel filtration and native gel electrophoresis we demonstrate that the majority of HHR23B is in a free, non-complexed form, and that a minor fraction is tightly associated with XPC. In contrast, we cannot detect any bound HHR23A. Thus the HHR23 proteins may have an additional function independent of XPC. The fractionation behaviour suggests that the non-bound forms of the HHR23 proteins are not necessary for the core of the NER reaction. Although both HHR23 proteins share a high level of overall homology, they migrate very differently on native gels, pointing to a difference in conformation. Gel filtration suggests the XPC-HHR23B heterodimer resides in a high MW complex. However, immunodepletion studies starting from repair-competent Manley extracts fall to reveal a stable association of a significant fraction of the HHR23 proteins or the XPC-HHR23B complex with the basal transcription/repair factor TFIIH, or with the ERCC1 repair complex. Consistent with a function in repair or DNA/chromatin metabolism, immunofluorescence studies show all XPC, HHR23B and (the free) HHR23A to reside in the nucleus.
van der Spek
P J
PJ
Department of Cell Biology and Genetics, Medical Genetic Centre, Erasmus University, Rotterdam, The Netherlands.
Eker
A
A
Rademakers
S
S
Visser
C
C
Sugasawa
K
K
Masutani
C
C
Hanaoka
F
F
Bootsma
D
D
Hoeijmakers
J H
JH
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002451:Cell Compartmentation; D002467:Cell Nucleus / Q000737:chemistry; D006224:Cricetinae; D004260:DNA Repair; D045643:DNA Repair Enzymes; D004268:DNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D011485:Protein Binding; D011994:Recombinant Proteins / Q000302:isolation & purification; D014983:Xeroderma Pigmentosum / Q000737:chemistry*
D004268:DNA-Binding Proteins; C513287:RAD23B protein, human; D011994:Recombinant Proteins; C086985:RAD23A protein, human; C086984:XPC protein, human; D045643:DNA Repair Enzymes