Purification of the yeast Slx5-Slx8 protein complex and characterization of its DNA-binding activity.
SLX5 and SLX8 encode RING-finger proteins that were previously identified based on their requirement for viability in yeast cells lacking Sgs1 DNA helicase. Slx5 and Slx8 proteins are known to be required for genome stability and to physically interact in yeast extracts; however, their biochemical functions are unknown. To address this question we purified and characterized recombinant Slx5 and Slx8 proteins. Here we show that Slx5 and Slx8 form a heterodimeric complex with double-stranded DNA (dsDNA)-binding activity. Individually, only the Slx8 subunit displays this activity. Structure-function studies indicate that the DNA-binding activity requires only the N-terminal 160 amino acids of Slx8, but not its C-terminal RING-finger domain. Alleles of SLX8 that express the RING-finger domain alone show almost complete complementation in yeast indicating that this DNA-binding domain is not essential for this in vivo function. Consistent with these findings we show that Slx5 immunolocalizes to the nucleus and that a portion of the Slx8 protein co-fractionates with chromatin. These results suggest that Slx5-Slx8 may act directly on DNA to promote genome stability.
Yang
Litao
L
Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, NJ 08854, USA.
Mullen
Janet R
JR
Brill
Steven J
SJ
D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000378:metabolism; D011994:Recombinant Proteins / Q000032:analysis / Q000302:isolation & purification; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity; D044767:Ubiquitin-Protein Ligases
D004268:DNA-Binding Proteins; D021122:Protein Subunits; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D004247:DNA; C515574:Slx8 protein, S cerevisiae; D044767:Ubiquitin-Protein Ligases; C515573:Slx5 protein, S cerevisiae