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J. Biol. Chem., Vol. 283, Issue 19, 12730-12735, May 9, 2008

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Identification of Regulatory Factor X as a Novel Mismatch Repair Stimulatory Factor^*

Yanbin Zhang¹, Fenghua Yuan¹, Daojing Wang, Liya Gu, and Guo-Min Li²

From the Department of Toxicology, University of Kentucky College of Medicine, Lexington, Kentucky 40536 and the Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720

This report describes the identification and purification of a novel mismatch repair stimulatory factor from HeLa cell extracts. This activity copurifies with a proliferating cell nuclear antigen-dependent 5 ' 3 ' DNA excision activity during several purification steps but is resolved from the excision activity during gel filtration chromatography using Sephacryl S-300. After purification to near homogeneity, the stimulatory factor is associated with three polypeptides with apparent molecular masses of 68, 36, and 30 kDa. Peptide sequencing analysis by tandem mass spectrometry identified the stimulatory factor as the heterotrimeric regulatory factor X (RFX) complex, which regulates transcription of the class II major histocompatibility complex by facilitating histone acetylation and is defective in the human hereditary immunodeficiency syndrome called bare lymphocyte syndrome. This conclusion was confirmed by the facts that purified recombinant RFX stimulates mismatch repair in an in vitro reconstituted mismatch repair system and that depletion of RFX from nuclear extracts or RFX knockdown in cells reduces mismatch repair activity. As expected, RFX knockdown cells display instability in microsatellite sequences. The possible role of RFX in human MMR repair is discussed.

Received for publication, January 17, 2008 , and in revised form, February 28, 2008.

^* This work was supported, in whole or in part, by National Institutes of Health Grants CA115942 and GM072756 (to G.-M. L.) and CA104333 (to L. G.). This work was also supported by a grant from the DOE/NASA Low Dose Radiation Research Program (to D. W.). 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Table S1 and supplemental Figs. S1 and S2

¹ Present address: Dept. of Biochemistry & Molecular Biology, University of Miami School of Medicine, Miami, FL.

² Recipient of the James-Gardner Endowed Chair in Cancer Research. To whom correspondence should be addressed: Dept. of Toxicology, University of Kentucky College of Medicine, 1095 VA Dr., Lexington, KY 40536. Tel.: 859-257-7053; Fax: 859-323-1059; E-mail: gmli{at}uky.edu.