Zappulla David C, Maharaj Arindel S R, Connelly Jessica J, Jockusch Rebecca A, Sternglanz Rolf
Department of Biochemistry and Cellular Biology, Stony Brook University, Stony Brook, NY 11794, USA.
BMC Mol Biol. 2006 Nov 9;7:40. doi: 10.1186/1471-2199-7-40.
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.
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.
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.
通过筛选一个质粒文库，寻找在酿酒酵母中靶向HMR位点时能够导致基因沉默的蛋白质，我们先前报道了基于Rtt107/Esc4建立沉默染色质的能力而对其进行的鉴定。在本研究中，我们旨在确定Rtt107/Esc4靶向沉默的机制，并更多地了解其生物学功能。
Rtt107/Esc4的靶向沉默依赖于SIR基因，这些基因编码酵母沉默染色质的必需结构和酶成分。基于其序列，预测Rtt107/Esc4含有六个BRCT基序。这个基序最初是在人类乳腺癌抑癌基因BRCA1中发现的，是一个蛋白质相互作用结构域。Rtt107/Esc4的靶向沉默活性位于C端的两个BRCT基序内，并且在双杂交试验中该蛋白的这一区域与Sir3结合。删除RTT107/ESC4会导致对DNA损伤剂MMS以及羟基脲敏感。双杂交筛选表明，Rtt107/Esc4的N端BRCT基序与Slx4结合，Slx4是一种先前显示参与DNA修复且在缺乏DNA解旋酶Sgs1的菌株中生存所必需的蛋白质。与SLX基因一样，RTT107ESC4与SGS1发生遗传相互作用；esc4Δsgs1Δ突变体是可存活的，但表现出生长缓慢的表型以及协同的DNA修复缺陷。
Rtt107/Esc4通过不同的BRCT基序与沉默蛋白Sir3和DNA修复蛋白Slx4结合，从而提供了一座连接沉默染色质与DNA修复酶的桥梁。
