The role of Esc2 and DNA in Smc5/6-mediated Sgs1-Top3-Rmi1 sumoylation
Mutchler, Ashley S.
Deoxyribonucleic acid (DNA) is arguably one of the most important biological materials. DNA contains the genetic material required to make up an organism and can be referred to as the body’s instruction manual. The replication of DNA is essential for the growth and reproduction of cells and requires precise copying of the genome, which is challenged by many obstacles or damages such as DNA double-strand breaks (DSBs). There are several mechanisms to deal with DSBs and replication blockages, including homologous recombination (HR). HR leads to the formation of DNA intermediates such as Holliday junctions (HJ), which are processed by one of two mechanisms: resolution or dissolution. In double Holliday junction dissolution, Sgs1-Top3-Rmi1 (STR) complex in yeast creates non-crossover products, and this process is regulated by Mms21-Smc5/6 mediated sumoylation of STR in the S and G2 phases. While STR is known to work in conjunction with Smc5/6 and SUMO, the exact details and mechanisms of how Mms21-Smc5/6 mediated STR sumoylation is regulated remain vastly unknown. This dissertation seeks to further investigate the mechanism in which genome stability factor Esc2 acts to remove recombinational intermediates through regulating the Mms21-Smc5/6 mediated STR sumoylation. Here, we found that Esc2 specifically influences the Mms21 (SUMO E3) substrates associated with HJs or replication fork structures. Importantly, our results also showed that Esc2 enhances the sumoylation of STR complex both in vitro and in vivo, which is mediated by its SLD2 domain that interacts with Ubc9, and that this interaction is essential in the dissolution of HJ and genome stability. Therefore, we suggest that Esc2 is a SUMO E2 cofactor that facilitates the sumoylation of Mms21 substrate STR in HJ dissolution. In addition, we found that the mid-region (MR) of Esc2 with HJ and replication fork binding activity also contributes to Sgs1 sumoylation, but does so through a DNA-independent manner. We also studied how STR sumoylation is regulated by DNA intermediates. We found that Sgs1 binding to DNA promotes its sumoylation, and Sgs1 sumoylation is enhanced when Esc2 is present. Ultimately, our data showed that Esc2 and DNA stimulated the Smc5/6-mediated sumoylation of the STR complex, which promoted the dissolution of DNA intermediates to maintain genome stability. Our results set a foundation for future studies on other potential binding partners of Ubc9 to better understand the efficiency of sumoylation, and also if sumoylation of Sgs1 affects its other roles in HR, such as end resection or D-loop assembly, to deepen the understanding of how sumoylation regulation of STR affects genome maintenance.
Esc2, Homologous recombination, Sumoylation, Genome maintenance, DNA
Mutchler, A. S. (2022). <i>The role of Esc2 and DNA in Smc5/6-mediated Sgs1-Top3-Rmi1 sumoylation</i> (Unpublished dissertation). Texas State University, San Marcos, Texas.