Cul8/Rtt101 Forms a Variety of Protein Complexes That Regulate DNA Damage Response and Transcriptional Silencing

Chikashi Obuse,Emiko Noro,Satoru Mimura,Satoru Ishii,Tomoya Katsura,Tsuyoshi Yamaguchi,Takumi Kamura

doi:10.1074/jbc.m109.082107

Chikashi Obuse, Emiko Noro + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m109.082107

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Abstract

The budding yeast, Saccharomyces cerevisiae, has three cullin proteins, which act as platforms for Cullin-based E3 ubiquitin ligases. Genetic evidence indicates that Cul8, together with Mms1, Mms22, and Esc4, is involved in the repair of DNA damage that can occur during DNA replication. Cul8 is thought to form a complex with these proteins, but the composition and the function of Cul8-based E3 ubiquitin ligases remain largely uncharacterized. Herein, we report a comprehensive biochemical analysis of Cul8 complexes. Cul8 was found to form a Cul8-Mms1-Mms22-Esc4 complex under physiological conditions, with Mms1 bridging Cul8 and Mms22 and Mms22 bridging Mms1 and Esc4. Domain analysis demonstrated that the N-terminal region of Mms1 and the C-terminal region of Mms22 are required for the Mms1-Mms22 interaction, whereas the N-terminal region of Mms22 is required for the Mms22-Esc4 interaction. We also found other Cul8-Mms1-binding proteins Ctf4, Esc2, and Orc5 using yeast two-hybrid screening. Esc4 and Ctf4 bound to Mms22 directly and bound to Cul8-Mms1 in the presence of Mms22, whereas Esc2 and Orc5 interacted with both Cul8 and Mms1, independently. We found that Cul8, Mms1, and Mms22 participated in the regulation of transcriptional silencing of yeast telomeres. These results suggest that Cul8-Mms1, as part of various protein complexes, is involved in the regulation of chromatin metabolism.

Highlights

Genotype SourceYLD70 YSM229 YSI25 YSI29 YSI38 YSI34 YTY143 YTY144 YTY141YTY080 YSI8 YSI41 YTK86 YTK87 YTK88 YTY106 YSI27 YTY083 YTY084 YTY089 YTY091 YTY055 YTY085 YSI2 YTY086 YTY048 YTY088 YTY056 YTY087 YTY127 YSH313 YTY121 YTY122 YTY123 YTY125 YTY124 YTY126W303-1a W303-1a W303-1a W303-1a W303-1a W303-1a W303-1a W303-1aW303-1a W303-1a W303-1aL40 L40 L40 W303-1a W303-1a W303-1a W303-1a W303-1a W303-1a W303-1a W303-1a W303-1a W303-1a W303-1a W303-1a W303-1a W303-1a W303-1aYSH313 YSH313 YSH313 YSH313 YSH313 YSH313
Using a yeast strain in which Cul8, Mms1, and Mms22 were epitopetagged with Myc, HA, and Pk, respectively, we found that endogenous Cul8 interacted with endogenous Mms1 and
Cul8/Rtt101 is a cullin protein reportedly involved in the regulation of DNA replication subsequent to DNA damage [4, 5, 11]

Summary

The abbreviations used are

E1, ubiquitin-activating enzyme; E2, ubiquitinconjugating enzyme; E3, ubiquitin ligase; SCF, Skp1-cullin-F-box protein; YPD, yeast extract-peptone-dextrose; MMS, methyl methanesulfonate; HA, hemagglutinin; Pk, small epitope (Pk) present on the P and V proteins of the paramyxovirus of simian virus; HSV, Herpes simplex virus of glycoprotein D. The parental strains were W303-1a: MATa leu112 trp can100 ura ade his and L40: MATa his3⌬ 200 trp901 leu2-3112 ade LYS2::(4lexAop-HIS3) URA3::(8lexAop-lacZ)GAL

Background

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION