Abstract
hHR23B is one of two human homologs of the Saccharomyces cerevisiae nucleotide excision repair (NER) gene product RAD23 and a component of a protein complex that specifically complements the NER defect of xeroderma pigmentosum group C (XP-C) cell extracts in vitro. Although a small proportion of hHR23B is tightly complexed with the XP-C responsible gene product, XPC protein, a vast majority exists as an XPC-free form, indicating that hHR23B has additional functions other than NER in vivo. Here we demonstrate that the human NER factor hHR23B as well as another human homolog of RAD23, hHR23A, interact specifically with S5a, a subunit of the human 26 S proteasome using the yeast two-hybrid system. Furthermore, hHR23 proteins were detected with S5a at the position where 26 S proteasome sediments in glycerol gradient centrifugation of HeLa S100 extracts. Intriguingly, hHR23B showed the inhibitory effect on the degradation of (125)I-lysozyme in the rabbit reticulocyte lysate. hHR23 proteins thus appear to associate with 26 S proteasome in vivo. From co-precipitation experiments using several series of deletion mutants, we defined the domains in hHR23B and S5a that mediate this interaction. From these results, we propose that part of hHR23 proteins are involved in the proteolytic pathway in cells.
Highlights
HHR23B is one of two human homologs of the Saccharomyces cerevisiae nucleotide excision repair (NER) gene product RAD23 and a component of a protein complex that complements the NER defect of xeroderma pigmentosum group C (XP-C) cell extracts in vitro
A small proportion of hHR23B is tightly complexed with the XP-C responsible gene product, XPC protein, a vast majority exists as an XPC-free form, indicating that hHR23B has additional functions other than NER in vivo
Using reconstituted cell-free NER reactions, we further showed that both hHR23 proteins enhance the repair activity of XPC, suggesting a possible functional interchangeability between the two RAD23 homologs (3)
Summary
HHR23B is one of two human homologs of the Saccharomyces cerevisiae nucleotide excision repair (NER) gene product RAD23 and a component of a protein complex that complements the NER defect of xeroderma pigmentosum group C (XP-C) cell extracts in vitro. By deletion and truncation analysis of recombinant hHR23B protein, the third domain has been recently found to be responsible for binding the XPC protein (6) These deletion studies revealed that the identified XPC-binding domain of hHR23B is necessary and largely sufficient for the hHR23B NER function in vitro: the Ub-like sequence and two copies of the ubiquitin-associated domains appear to be dispensable for the core part of NER. It is conceivable that hHR23B, as well as other members of this class of proteins, may be associated with ubiquitin metabolic pathways outside the context of the core NER machinery In agreement with this idea, hHR23B exists in vivo in a large excess over XPC (7, 8), suggesting it has extra functions without XPC
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