Purification and Characterization of the XPF-ERCC1 Complex of Human DNA Repair Excision Nuclease

Chi-Hyun Park,Tadayoshi Bessho,Tsukasa Matsunaga,Aziz Sancar

doi:10.1074/jbc.270.39.22657

Chi-Hyun Park, Tadayoshi Bessho + Show 2 more

Open Access

https://doi.org/10.1074/jbc.270.39.22657

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Abstract

A complex, which consists of ERCC1 (38 kDa) and a 112-kDa protein, was purified from HeLa cells to homogeneity. This complex complemented the nucleotide excision repair defects of rodent ERCC-1, ERCC-4, and human XP-F mutant cell-free extracts, indicating that the 112-kDa protein is XPF/ERCC4 and providing direct biochemical evidence that XPF and ERCC4 are identical. The XPF/ERCC4-ERCC1 complex has an endonuclease activity with preference for single-stranded DNA and a single-stranded region of duplex DNA with a "bubble" structure. This complex also nicks supercoiled DNA weakly, and this nicking activity is stimulated by human replication protein A when the DNA contains UV damage.

Highlights

Purification of XPF/ERCC4 in the Complex with ERCC1 from HeLa Cell-free Extract—The ERCC1-containing complex was purified from 400 liters of HeLa cell culture using procedures modified from Mu et al [7] in which an XPA-affinity chromatography is the major purification step, since XPA protein binds to ERCC1 protein with high affinity [6, 9]
The affinity-purified complex was purified further through two additional columns, DEAE-Sepharose followed by heparin-agarose, and only two polypeptides were detected in the final fraction by silver staining (Fig. 1, lane 1)
It was recently reported that partially purified ERCC1 fractions complemented both XP-F and ERCC-4 CFEs as well as ERCC-1 CFE [7, 8], raising the possibility that the 112-kDa protein, which makes a complex with ERCC1, is XPF and/or ERCC4

Summary

Introduction

1 The abbreviations used are: XP, xeroderma pigmentosum; CFE, cell-free extract; ERCC, excision repair cross-complementing; RPA, replication protein A; bp, base pair(s); SSB, single-stranded DNA binding. Endonuclease Assay—Covalently closed single-stranded M13 mp19 DNA (125 ng) or covalently closed supercoiled double-stranded pMLU112 DNA (100 ng) was incubated at 37 °C for the indicated time in 15 ␮l of nuclease reaction buffer (25 mM HEPES-KOH, pH 7.9, 25 mM KCl, 3 mM MgCl2, 1 mM dithiothreitol, 120 ␮g/ml bovine serum albumin, and 6.5% glycerol) with the XPF-ERCC1 complex.

Results

Conclusion