HHR23B, a human Rad23 homolog, stimulates XPC protein in nucleotide excision repair in vitro.
A protein complex which specifically complements defects of XP-C cell extracts in vitro was previously purified to near homogeneity from HeLa cells. The complex consists of two tightly associated proteins: the XPC gene product and HHR23B, one of two human homologs of the Saccharomyces cerevisiae repair gene product Rad23 (Masutani et al., EMBO J. 13:1831-1843, 1994). To elucidate the roles of these proteins in "genome-overall" repair, we expressed the XPC protein in a baculovirus system and purified it to near homogeneity. The recombinant human XPC (rhXPC) protein exhibited a high level of affinity for single-stranded DNA and corrected the repair defect in XP-C whole-cell extracts without extra addition of recombinant HHR23B (rHHR23B) protein. However, Western blot (immunoblot) experiments revealed that XP-C cell extracts contained excess endogenous HHR23B protein, which might be able to form a complex upon addition of the rhXPC protein. To investigate the role of HHR23B, we fractionated the XP-C cell extracts and constructed a reconstituted system in which neither endogenous XPC nor HHR23B proteins were present. In this assay system, rhXPC alone weakly corrected the repair defect, while significant enhancement of the correcting activity was observed upon coaddition of rHHR23B protein. Stimulation of XPC by HHR23B was found with simian virus 40 minichromosomes as well as with naked plasmid DNA and with UV- as well as N-acetoxy-2- acetylfluorene-induced DNA lesions, indicating a general role of HHR23B in XPC functioning in the genome-overall nucleotide excision repair subpathway.
Sugasawa
K
K
The Institute of Physical and Chemical Research (RIKEN), Saitama, Japan.
Masutani
C
C
Uchida
A
A
Maekawa
T
T
van der Spek
P J
PJ
Bootsma
D
D
Hoeijmakers
J H
JH
Hanaoka
F
F
D000818:Animals; D001483:Base Sequence; D004260:DNA Repair / Q000187:drug effects*; D045643:DNA Repair Enzymes; D004274:DNA, Recombinant / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017924:Nucleopolyhedroviruses / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013539:Simian virus 40 / Q000235:genetics; D018411:Spodoptera; D014983:Xeroderma Pigmentosum / Q000473:pathology
D004274:DNA, Recombinant; D004268:DNA-Binding Proteins; C513287:RAD23B protein, human; D011993:Recombinant Fusion Proteins; C086985:RAD23A protein, human; C086984:XPC protein, human; D045643:DNA Repair Enzymes