(WT) Wild type. structures. Our targeted screen found that Esc2 promotes the sumoylation of a Holliday junction dissolution complex and specific replisome proteins. Esc2 does not elicit these effects via stable interactions with substrates or their common SUMO E3. Rather, Mouse monoclonal to CD44.CD44 is a type 1 transmembrane glycoprotein also known as Phagocytic Glycoprotein 1(pgp 1) and HCAM. CD44 is the receptor for hyaluronate and exists as a large number of different isoforms due to alternative RNA splicing. The major isoform expressed on lymphocytes, myeloid cells and erythrocytes is a glycosylated type 1 transmembrane protein. Other isoforms contain glycosaminoglycans and are expressed on hematopoietic and non hematopoietic cells.CD44 is involved in adhesion of leukocytes to endothelial cells,stromal cells and the extracellular matrix we show that a SUMO-like domain name of Esc2 stimulates sumoylation by exploiting a noncovalent SUMO binding site around the E2 enzyme. This role of Esc2 in sumoylation is required for Holliday junction clearance and genome stability. Our findings thus suggest that Esc2 acts as a SUMO E2 cofactor at distinct DNA structures to promote the sumoylation of specific substrates and genome maintenance. and mutants accumulate HJ structures upon MMS treatment that are resolved by the STR complex (Branzei et al. 2006; Mankouri et al. 2009; Sollier et al. 2009), we first examined STR sumoylation. Mms21-mediated STR sumoylation is usually thought to occur at HJs and contribute to HJ clearance (Bermdez-Lpez et al. 2016; Bonner et al. 2016). We found that in cells, all three STR subunits had reduced sumoylation levels upon MMS treatment (Fig. 1A), suggesting that this defect could underlie the elevated HJ levels in cells in this condition. Open Tenatoprazole in a separate window Physique 1. Esc2 promotes the sumoylation of a specific set of Mms21 substrates. (cells. Cells made up of His8-tagged SUMO were treated with 0.03% MMS for 2 h to induce STR sumoylation. Sumoylated proteins were isolated using Ni-NTA resins and examined by immunoblotting using antibodies recognizing the tag fused to the endogenous Sgs1, Top3, or Rmi1 to visualize sumoylated forms of the proteins (-S), which are indicated by lines next to the blots. Loading is shown by Ponceau S stain (stain). (WT) Wild type. Comparable methods for examining sumoylation and annotation of immunoblots are used in subsequent panels unless otherwise noted. (reduces the levels of mono-sumoylated form of Pol2 and di-sumoylated form of Mcm3. (cells maintain the sumoylation levels of Yku70 and Rfa1. Yku70 sumoylation was examined as in (as shown previously Tenatoprazole (Chung and Zhao 2015). (cells maintain the sumoylation levels for the subunits of three SMC complexes, namely cohesin, condensin, and the Smc5/6 complex. Sumoylation of Smc2 was examined as in (on Mms21-dependent sumoylation of DNA metabolism proteins. Results in show that reduces the sumoylation of proteins known to associate with HJ and replication fork structures but not those that mainly interact with ssDNA or dsDNA. Since Esc2 preferentially binds to HJs and replication fork structures (Urulangodi et al. 2015; Sebesta et al. 2017), we next examined two replisome proteins whose sumoylation is usually regulated by Mms21. Monosumoylation of the leading strand DNA polymerase Pol2, which was suggested to occur at replication forks, and the disumoylated form of the Mcm3 subunit of Tenatoprazole the replicative helicase are largely abolished in E3 mutants (Albuquerque et al. 2016; Meng et al. 2019; Winczura et al. 2019). We found that phenocopied the mutant in both cases (Fig. 1B). In contrast, cells were proficient for the sumoylation Tenatoprazole of Mms21 substrates located at other types of DNA structures (Fig. 1C,D). These include the dsDNA end binding protein Yku70, ssDNA binding protein Rfa1, and subunits of three SMC complexes that predominantly associate with dsDNA (Zhao and Blobel 2005; Takahashi et al. 2008; Whalen et al. 2020). In addition, cells maintained the sumoylation of Siz E3 substrates that bind to HJ, replication fork, or DNA flap structures (Supplemental Fig. S1ACC; Hoege et al. 2002; Stelter and Ulrich 2003; Sarangi et al. 2014; Talhaoui et al. 2018). Our data (summarized in Fig. 1E) suggest that, while Esc2 has a functional partnership with Mms21, it does not act as a general regulator of this Tenatoprazole E3; rather, Esc2 specifically contributes to the sumoylation of Mms21 substrates associating with HJs and replication fork structures. Query of Esc2 interactions with SUMO E2, SUMO E3, SUMO, and SUMO substrates Consistent with the above notion that Esc2 is not a general Mms21 regulator, we did not detect association of Esc2 with the Smc5/6 complex, of which Mms21 is an obligate subunit, either through coimmunoprecipitation or yeast two-hybrid assays (Fig. 2A,B). We also did not find evidence that Esc2 could bind to the SUMO substrates identified above or bridge their interactions with Mms21. Yeast two-hybrid tests did not reveal conversation of Esc2 with STR subunits, Pol2, or Mcm3 (Fig. 2B). Moreover, Esc2 did not coimmunoprecipitate with Sgs1 or Pol2, whereas the association of Sgs1 with Smc5 or Pol2 with its partner protein Dpb2 was detected (Fig. 2A,C; Supplemental Fig. S2A). Purified Esc2 did not show conversation with STR or Smc5/6 in vitro (Supplemental Fig. S2B,C). Finally, Esc2 loss did not affect Smc5 association with Sgs1 or Pol2 in vivo (Fig. 2D; Supplemental Fig. S2D). Open in a separate window Figure.
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