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Molecular and Cellular Biology, July 2004, p. 6362-6378, Vol. 24, No. 14
0270-7306/04/$08.00+0     DOI: 10.1128/MCB.24.14.6362-6378.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

The NEF4 Complex Regulates Rad4 Levels and Utilizes Snf2/Swi2-Related ATPase Activity for Nucleotide Excision Repair

Kerrington L. Ramsey, Joshua J. Smith, Arindam Dasgupta, Nazif Maqani, Patrick Grant, and David T. Auble*

Department of Biochemistry and Molecular Genetics, University of Virginia Health System, Charlottesville, Virginia 22908-0733

Received 23 March 2004/ Accepted 20 April 2004

Nucleotide excision repair factor 4 (NEF4) is required for repair of nontranscribed DNA in Saccharomyces cerevisiae. Rad7 and the Snf2/Swi2-related ATPase Rad16 are NEF4 subunits. We report previously unrecognized similarity between Rad7 and F-box proteins. Rad16 contains a RING domain embedded within its ATPase domain, and the presence of these motifs in NEF4 suggested that NEF4 functions as both an ATPase and an E3 ubiquitin ligase. Mutational analysis provides strong support for this model. The Rad16 ATPase is important for NEF4 function in vivo, and genetic analysis uncovered new interactions between NEF4 and Rad23, a repair factor that links repair to proteasome function. Elc1 is the yeast homologue of a mammalian E3 subunit, and it is a novel component of NEF4. Moreover, the E2s Ubc9 and Ubc13 were linked to the NEF4 repair pathway by genetic criteria. Mutations in NEF4 or Ubc13 result in elevated levels of the DNA damage recognition protein Rad4 and an increase in ubiquitylated species of Rad23. As Rad23 also controls Rad4 levels, these results suggest a complex system for globally regulating repair activity in vivo by controlling turnover of Rad4.

* Corresponding author. Mailing address: Department of Biochemistry and Molecular Genetics, University of Virginia Health System, 1300 Jefferson Park Ave., Room 6213, Charlottesville, VA 22908-0733. Phone: (434) 243-2629. Fax: (434) 924-5069. E-mail: dta4n{at}virginia.edu.

Molecular and Cellular Biology, July 2004, p. 6362-6378, Vol. 24, No. 14
0022-538X/04/$08.00+0     DOI: 10.1128/MCB.24.14.6362-6378.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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