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

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.The ubiquitin/proteasome-dependent protein degradation system is a highly regulated and specific system for targeted protein degradation and is involved in virtually all of the biological processes in eukaryotes. In this system, the ubiquitin is conjugated to a substrate protein, and subsequent polyubiquitination of the substrate ultimately creates a recognition site for the 26 S proteasome. The specificity of protein degradation is determined by the recognition of specific substrates at specific times.Protein ubiquitination involves three enzymes: E1, 3 E2, and the ubiquitin ligase E3. Only E3 ligases can directly recognize specific substrates, i.e. they determine substrate specificity. There are many different types of E3 ligases. One such E3 is the cullin-based ubiquitin ligase. The Skp1-cullin-F-box protein (SCF) complex is the most studied cullin-based E3 ubiquitin ligase (1, 2). SCF consists of the RING finger protein, Rbx1, cullin, Cul1/Cdc53, the adapter protein, Skp1, and a substrate recognition subunit, which is an F-box protein. Rbx1, Cul1, and Skp1 are constitutive subunits of SCF, but the F-box protein is a variable subunit; multiple F-box proteins are present in cells and respond to different substrates.Multiple and different cullins are found in different species, e.g. humans have six cullins, namely Cul1 through Cul5 and Cul7. Each cullin has specific adapter proteins and substrate recognition substrates, such as Elongin BC and Cul2-box proteins for Cul2, BTB (Bric-a-brac, Tramtrack, Broad complex) proteins for Cul3, Ddb1 and Cul4-box proteins for Cul4, Elongin BC and SOCS box proteins for Cul5, and Skp1 and F-box...

Replisome Function During Replicative Stress Is Modulated by Histone H3 Lysine 56 Acetylation Through Ctf4

“…Finally, our data strongly suggest that this effect is dependent upon an interaction between Ctf4 and Mms22. Similarly to ctf4D, deletion of MRC1 induces uncoupling between helicase and polymerase (Tanaka et al 2009;Mimura et al 2010;Vaisica et al 2011). In accord with this notion, we found that the replication function of MRC1 is also strongly deleterious for cells experiencing constitutive replicative damages in the absence of a functional H3K56ac pathway.…”

“…In the absence of RRM3, chromosome breaks occur at discrete fork pause sites at specific genomic locations (Ivessa et al 2003). A number of studies indicate that the DNA breaks generated in rrm3D cells affect cell viability in the absence of the so-called "H3K56 acetylation pathway" that comprises ASF1, RTT109, RTT101, MMS1, and MMS22 (Tong et al 2004;Luke et al 2006;Pan et al 2006;Collins et al 2007;Duro et al 2008;Roberts et al 2008;Zaidi et al 2008;Costanzo et al 2010;Koh et al 2010;Mimura et al 2010).In S. cerevisiae, H3K56 localizes at the DNA entry and exit points of a nucleosome (Masumoto et al 2005;Ozdemir et al 2005;Xu et al 2005). H3K56 is transiently acetylated during the S phase of the cell cycle and after DNA damage and is rapidly de-acetylated by the action of the sirtuins Hst3 and Hst4, when cells enter the transition between G2 and M phases and after DNA repair (Masumoto et al 2005;Xu et al 2005).…”

“…CTF4 is not essential for budding yeast viability (Miles and Formosa 1992), but its deletion greatly sensitizes cells to DNA replication drugs (Ogiwara et al 2007). Mechanistically, Ctf4 is required for coordination between DNA unwinding and synthesis, and it also stabilizes polymerase-a at the replication forks (Gambus et al 2009;Tanaka et al 2009;Mimura et al 2010). Among various partners, Ctf4 interacts with an F-box protein Dia2 involved in the regulation of DNA replication (Mimura et al 2009) and with Mms22, an adaptor protein of the Cul4(Ddb1)-like E3 ubiquitin ligase complex (Gambus et al 2009;Mimura et al 2009Mimura et al , 2010.…”

“…Mechanistically, Ctf4 is required for coordination between DNA unwinding and synthesis, and it also stabilizes polymerase-a at the replication forks (Gambus et al 2009;Tanaka et al 2009;Mimura et al 2010). Among various partners, Ctf4 interacts with an F-box protein Dia2 involved in the regulation of DNA replication (Mimura et al 2009) and with Mms22, an adaptor protein of the Cul4(Ddb1)-like E3 ubiquitin ligase complex (Gambus et al 2009;Mimura et al 2009Mimura et al , 2010. The latter also includes Mms1 and cullin Rtt101, both crucial for maintaining replisome integrity in hydroxyurea and therefore for efficient recovery from replication stress (Luke et al 2006;Duro et al 2008;Zaidi et al 2008;Gambus et al 2009;Mimura et al 2010;Vaisica et al 2011).…”

“…Among various partners, Ctf4 interacts with an F-box protein Dia2 involved in the regulation of DNA replication (Mimura et al 2009) and with Mms22, an adaptor protein of the Cul4(Ddb1)-like E3 ubiquitin ligase complex (Gambus et al 2009;Mimura et al 2009Mimura et al , 2010. The latter also includes Mms1 and cullin Rtt101, both crucial for maintaining replisome integrity in hydroxyurea and therefore for efficient recovery from replication stress (Luke et al 2006;Duro et al 2008;Zaidi et al 2008;Gambus et al 2009;Mimura et al 2010;Vaisica et al 2011).The Rrm3 helicase travels with the replication fork and facilitates the progression of replication forks through nonhistone protein-DNA complexes throughout the genome (Azvolinsky et al 2009;Fachinetti et al 2010). In the absence of RRM3, chromosome breaks occur at discrete fork pause sites at specific genomic locations (Ivessa et al 2003).…”