Functional analysis of the DNA-stimulated ATPase domain of yeast SW12/SNF2

Abstract

The yeast SW12/SNF2 polypeptide is a subunit of the SWI/SNF protein complex that is required for many transcriptional activators to function in a chromatin context. SW12 is believed to be the founding member of a new subfamily of DNA-stimulated ATPases/DNA helicases that includes proteins that function in DNA repair (RAD5, RAD16, ERCC6), recombination (RAD54), transcription (MOT1, ISWI, brm, BRG1, hBRM) and cell cycle control (STH1). We have created a set of 16 mutations within the SW12 ATPase domain and have analyzed the functional consequences of these mutations in vivo. We have identified residues within each of the seven ATPase motifs that are required for SW12 function. We have also identified crucial residues that are interspersed between the known ATPase motifs. In contrast, we identify other highly conserved residues that appear to be dispensable for SW12 function. We also find that single amino acid changes in ATPase motifs IV and VI lead to a dominant negative phenotype. None of the 12 SW12 mutations that disrupt SW12 activity in vivo alter the assembly of the SWI/SNF complex. These studies provide an invaluable framework for biochemical analysis of the SW12 ATPase and for functional analysis of other SW12 family members.