Rtt107/Esc4 binds silent chromatin and DNA repair proteins using different BRCT motifs.
BACKGROUND
By screening a plasmid library for proteins that could cause silencing when targeted to the HMR locus in Saccharomyces cerevisiae, we previously reported the identification of Rtt107/Esc4 based on its ability to establish silent chromatin. In this study we aimed to determine the mechanism of Rtt107/Esc4 targeted silencing and also learn more about its biological functions.


RESULTS
Targeted silencing by Rtt107/Esc4 was dependent on the SIR genes, which encode obligatory structural and enzymatic components of yeast silent chromatin. Based on its sequence, Rtt107/Esc4 was predicted to contain six BRCT motifs. This motif, originally identified in the human breast tumor suppressor gene BRCA1, is a protein interaction domain. The targeted silencing activity of Rtt107/Esc4 resided within the C-terminal two BRCT motifs, and this region of the protein bound to Sir3 in two-hybrid tests. Deletion of RTT107/ESC4 caused sensitivity to the DNA damaging agent MMS as well as to hydroxyurea. A two-hybrid screen showed that the N-terminal BRCT motifs of Rtt107/Esc4 bound to Slx4, a protein previously shown to be involved in DNA repair and required for viability in a strain lacking the DNA helicase Sgs1. Like SLX genes, RTT107ESC4 interacted genetically with SGS1; esc4Delta sgs1Delta mutants were viable, but exhibited a slow-growth phenotype and also a synergistic DNA repair defect.


CONCLUSIONS
Rtt107/Esc4 binds to the silencing protein Sir3 and the DNA repair protein Slx4 via different BRCT motifs, thus providing a bridge linking silent chromatin to DNA repair enzymes.
Zappulla
David C
DC
Department of Biochemistry and Cellular Biology, Stony Brook University, Stony Brook, NY 11794, USA. david.zappulla@gmail.com <david.zappulla@gmail.com>
Maharaj
Arindel S R
AS
Connelly
Jessica J
JJ
Jockusch
Rebecca A
RA
Sternglanz
Rolf
R
D020816:Amino Acid Motifs / Q000235:genetics*; D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D002843:Chromatin / Q000378:metabolism*; D004260:DNA Repair; D004706:Endodeoxyribonucleases / Q000235:genetics / Q000378:metabolism; D020868:Gene Silencing; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D038281:Silent Information Regulator Proteins, Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D020798:Two-Hybrid System Techniques
D002843:Chromatin; D009687:Nuclear Proteins; C501265:RTT107 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D038281:Silent Information Regulator Proteins, Saccharomyces cerevisiae; D004706:Endodeoxyribonucleases; C505436:SLX4 protein, S cerevisiae