Two human homologs of Rad23 are functionally interchangeable in complex formation and stimulation of XPC repair activity.
XPC-hHR23B protein complex is specifically involved in nucleotide excision repair (NER) of DNA lesions on transcriptionally inactive sequences as well as the nontranscribed strand of active genes. Here we demonstrate that not only highly purified recombinant hHR23B (rhHR23B) but also a second human homolog of the Saccharomyces cerevisiae Rad23 repair protein, hHR23A, stimulates the in vitro repair activity of recombinant human XPC (rhXPC), revealing functional redundancy between these human Rad23 homologs. Coprecipitation experiments with His-tagged rhHR23 as well as sedimentation velocity analysis showed that both rhHR23 proteins in vitro reconstitute a physical complex with rhXPC. Both complexes were more active than free rhXPC, indicating that complex assembly is required for the stimulation. rhHR23B was shown to stimulate an early stage of NER at or prior to incision. Furthermore, both rhHR23 proteins function in a defined NER system reconstituted with purified proteins, indicating direct involvement of hHR23 proteins in the DNA repair reaction via interaction with XPC.
Sugasawa
K
K
Department of Cell Biology and Genetics, Medical Genetic Centre, Erasmus University, Rotterdam, The Netherlands. sugasawa@postman.riken.go.jp
Ng
J M
JM
Masutani
C
C
Maekawa
T
T
Uchida
A
A
van der Spek
P J
PJ
Eker
A P
AP
Rademakers
S
S
Visser
C
C
Aboussekhra
A
A
Wood
R D
RD
Hanaoka
F
F
Bootsma
D
D
Hoeijmakers
J H
JH
D004260:DNA Repair / Q000502:physiology*; D045643:DNA Repair Enzymes; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D066298:In Vitro Techniques; D046911:Macromolecular Substances; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism; D014983:Xeroderma Pigmentosum / Q000235:genetics / Q000378:metabolism
D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; C513287:RAD23B protein, human; D011994:Recombinant Proteins; C086985:RAD23A protein, human; C086984:XPC protein, human; D045643:DNA Repair Enzymes