Overproduction, Purification, and Characterization of the XPC Subunit of the Human DNA Repair Excision Nuclease

Joyce T Reardon,David Mu,Aziz Sancar

doi:10.1074/jbc.271.32.19451

Joyce T Reardon, David Mu + Show 1 more

Open Access

https://doi.org/10.1074/jbc.271.32.19451

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Abstract

Xeroderma pigmentosum complementation group C gene (XPC) encodes a protein of 125 kDa which is present in a tight complex with a 58-kDa protein encoded by the human homolog of the yeast RAD23 gene, HHR23B (Masutani, C., Sugasawa, K., Yanagisawa, J., Sonoyama, T., Ui, M., Enomoto, T., Takio, K., Tanaka, K., van der Spek, P. J., Bootsma, D., Hoeijmakers, J. H. J., and Hanaoka, F.(1994) EMBO J. 13, 1831-1843). The XPC-HHR23B complex is required for excision of thymine dimers from DNA in a human excision nuclease system reconstituted from purified proteins. In order to understand the role of the XPC-HHR23B complex in excision repair, we have overexpressed each subunit alone and the heterodimer in heterologous systems, purified them, and characterized their biochemical properties. We find that both XPC and the heterodimer bind DNA with high affinity and UV-damaged DNA with slightly higher preference. Surprisingly, we find that the XPC subunit alone is sufficient for reconstitution of the human excision nuclease and that the HHR23B subunit has no detectable effect on the excision activity of the reconstituted system.

Highlights

Nucleotide excision repair is a general repair system which is suited for removing bulky DNA lesions such as thymine dimers and cisplatin-d(GpG) diadducts (Friedberg et al, 1995; Sancar, 1996)
Upon purification of Xeroderma pigmentosum complementation group C gene (XPC) complementing activity, it was found that the 125-kDa protein encoded by XPC (Legerski and Peterson, 1992; Masutani et al, 1994) was in a complex with a protein of 58 kDa which is highly homologous to the yeast excision repair protein encoded by the RAD23 gene (Watkins et al, 1993; Masutani et al, 1994)
The yeast RAD23 gene belongs to the RAD3 epistasis group, whose members participate in excision repair

Summary

Introduction

Nucleotide excision repair is a general repair system which is suited for removing bulky DNA lesions such as thymine dimers and cisplatin-d(GpG) diadducts (Friedberg et al, 1995; Sancar, 1996). Proteins encoded by seven XP genes, XPA through XPG, are required for the dual incision (excision) step of nucleotide excision repair. XPC and HHR23B appear to be tightly bound and the final purification step for XPC yields these two proteins in 1:1 stoichiometry (Masutani et al, 1994; Aboussekhra et al, 1995; Mu et al, 1996), there was no genetic or biochemical data from mammalian systems indicating that HHR23B is involved in excision repair. We found that XPC and the XPC-HHR23B complex bind to DNA nonspecifically and with relatively high affinity and to UV-damaged DNA with slightly higher affinity. Both forms of XPC are capable of complementing cell-free extracts of XP-C mutants and are active in reconstituting excision nuclease activity in a completely defined system. We conclude that with naked DNA under our experimental conditions HHR23B does not play a direct role in excision repair

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