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J. Biol. Chem., Vol. 280, Issue 31, 28644-28652, August 5, 2005

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Biophysical Characterization of the Interaction Domains and Mapping of the Contact Residues in the XPF-ERCC1 Complex^*

Yun-Jeong Choi, Kyoung-Seok Ryu, Yun-Mi Ko, Young-Kee Chae¶, Jeffrey G. Pelton||, David E. Wemmer||**, and Byong-Seok Choi

From the Department of Chemistry and National Creative Research Initiative Center, Korea Advanced Institute of Science and Technology, 373-1 Guseong-dong, Yuseong-gu, Daejon 305-701, Korea, the ^¶Department of Applied Chemistry, Sejong University, 98 Gunja-Dong, Gwangjin-Gu, Seoul 143-747, Korea, and ^||Physical Biosciences Division, Lawrence Berkeley National Laboratory and Department of Chemistry, University of California, Berkeley, California 94720

XPF and ERCC1 exist as a heterodimer to be stable and active in cells and catalyze DNA cleavage on the 5'-side of a lesion during nucleotide excision repair. To characterize the specific interaction between XPF and ERCC1, we expressed the human ERCC1 binding domain of XPF (XPF-EB) and the XPF binding domain of ERCC1 (ERCC1-FB) in Escherichia coli. Milligram quantities of a heterodimer were characterized with gel filtration chromatography, an Ni²⁺-NTA binding assay, and analytical ultracentrifugation. Cross-linking experiments at high salt concentrations revealed that XPF interacts with ERCC1 mainly through hydrophobic interactions. XPF-EB was also shown to homodimerize in the absence of ERCC1. NMR cross-saturation methods were applied to map the residues involved in formation of the XPF-EB·XPF-EB homodimer and the XPF-EB·ERCC1-FB heterodimer. Helix H3 and the C-terminal region of XPF-EB were either within or in close proximity to the homodimer interface, whereas the ERCC1-FB binding site of XPF-EB was distributed across helix H1, a small part of H2, H3, and the C-terminal region, most of which exhibited large changes in chemical shift upon ERCC1 binding. The XPF-EB heterodimeric interface is larger than the XPF-EB homodimeric one, which could explain why XPF has a stronger affinity for ERCC1 than for a second molecule of XPF. The XPF binding sites of ERCC1 were located in helices H1 and H3 and in the C-terminal region, similar to the involved surface of XPF. We used cross-saturation data and the crystal structure of related proteins to model the two complexes.

Received for publication, January 31, 2005 , and in revised form, April 25, 2005.

^* This work was supported in part by the Creative Research Initiative Program from the Ministry of Science and Technology, Korea. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Supported in part by the BK21 project.

^** To whom correspondence may be addressed. Tel.: 510-486-4318; Fax: 510-486-6059; E-mail: dewemmer{at}lbl.gov.

To whom correspondence may be addressed. Tel.: 82-42-869-2868; Fax: 82-42-869-2810; E-mail: byongseok.choi{at}kaist.ac.kr.

This article has been cited by other articles:

				D. Yu, X. Zhang, J. Liu, P. Yuan, W. Tan, Y. Guo, T. Sun, D. Zhao, M. Yang, J. Liu, et al. Characterization of Functional Excision Repair Cross-Complementation Group 1 Variants and Their Association with Lung Cancer Risk and Prognosis Clin. Cancer Res., May 1, 2008; 14(9): 2878 - 2886. [Abstract] [Full Text] [PDF]