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

Abstract

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.

We are grateful to Brehon Laurent, Dan Gietz, Linda Hyman, Chris Koth, Mike Tyers, Mark Goebl, Mark Hochstrasser, Mary Ann Osley, Cecile Pickart, Alex Varshavsky, and Mitch Smith for the plasmids and yeast strains used in this study. We are also grateful to Justin Reese for advice on sequence analysis, Mitch Smith for critically reading the manuscript, Sarah Juedes for help with plasmid preparation and strain construction, Rick Wood and Cecile Pickart for advice and encouragement, and members of the Auble lab for helpful discussions.

This work was supported by grants from the NIH (GM55763), March of Dimes, and Kincaid Charitable Trust and an Institutional Research Award from the American Cancer Society to D.T.A.