Browsing by Subject "Chromatids"
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Item Cohesin, the SMC5/6 Complex and SUMO in DNA Repair(2013-01-17) Wu, Nan; Yu, HongtaoDNA double-strand breaks (DSBs) fuel cancer-driving chromosome translocations. Two related structural maintenance of chromosomes (Smc) complexes, cohesin and Smc5/6, promote DSB repair through sister-chromatid homologous recombination (SCR). Our results show that the Smc5/6 subunit Mms21 sumoylates multiple lysines of the cohesin subunit Scc1. Mms21 promotes cohesin-dependent SUMO accumulation at laser-induced DNA damage sites in S/G2 human cells. Cells expressing the non-sumoylatable Scc1 mutant (15KR) maintain sister-chromatid cohesion during mitosis, but are defective in SCR and sensitive to ionizing radiation (IR). Scc1 15KR is recruited to DNA damage sites. Depletion of Wapl, a negative cohesin regulator, rescues SCR defects of Mms21-deficient or Scc1 15KR-expressing cells. Expression of the acetylation-mimicking Smc3 mutant does not bypass the requirement for Mms21 in SCR. We propose that Scc1 sumoylation by Mms21 promotes SCR by antagonizing Wapl at a step after cohesin loading at DSBs and in a way not solely dependent on Smc3 acetylation. Our results establish a new posttranslational regulatory mechanism of cohesin during DNA repair, and reveal both conserved principles and organism-specific features in cohesin regulation during sister-chromatid recombinationItem Regulation of Sister Chromatid by the Acetyltransferase Naa50(2016-07-25) Rong, Ziye; Seemann, Joachim; Yu, Hongtao; Li, Bing; Olson, Eric N.During the cell cycle, sister-chromatid cohesion tethers sister chromatids together from S phase to the metaphase-anaphase transition and ensures accurate chromosome segregation of chromatids into daughter cells. N-terminal acetylation is one of the most prevalent protein covalent modifications in eukaryotes and is mediated by a family of N-terminal acetyltransferases (NAT). Naa50 (also called San or NatE) has previously been shown to play a role in sister-chromatid cohesion in metazoans. The mechanism by which Naa50 contributes to cohesion is not understood, however. Here, I show that depletion of Naa50 in HeLa cells weakens the interaction between cohesin and its positive regulator sororin and causes cohesion defects in interphase, consistent with a role of Naa50 in cohesion establishment or maintenance. Strikingly, co-depletion of NatA, a heterodimeric NAT complex that physically interacts with Naa50, rescues the sister-chromatid cohesion defects and the resulting mitotic arrest caused by Naa50 depletion, indicating that NatA and Naa50 play antagonistic roles in cohesion. Purified recombinant NatA and Naa50 do not affect each other's NAT activity in vitro. Because NatA and Naa50 exhibit distinct substrate specificity, I propose that they modify different effectors and regulate sister-chromatid cohesion in opposing ways.Item Regulation of Sister-Chromatid Cohesion(2017-10-17) Zheng, Ge; Burma, Sandeep; Yu, Hongtao; Mendell, Joshua T.; Tu, BenjaminOrderly execution of two critical events during the cell cycle--DNA replication and chromosome segregation--ensures the stable transmission of genetic materials. The cohesin complex physically connects sister chromatids during DNA replication in a process termed sister-chromatid cohesion. Timely establishment and dissolution of sister-chromatid cohesion is a prerequisite for accurate chromosome segregation, and is tight regulated by the cell cycle machinery and cohesin-associated proteins. Errors in this process can lead to aneuploidy and promote tumorigenesis. Research in this dissertation has provided several key insights into the regulation of sister-chromatid cohesion during the mitotic cell cycle. First, we report the crystal structure and functional characterization of human Wapl, a key negative regulator of cohesin that promotes cohesin release from chromatin. Our results indicate that Wapl-mediated cohesin release from chromatin requires extensive physical contacts between Wapl and multiple cohesin subunits. Second, we have determined the crystal structure of human SA2-Scc1 cohesin subcomplex, which is the interaction hub for cohesin regulators. Further biochemical and functional analyses reveal the direct competition between Wapl and the cohesion protector Sgo1 for binding to a conserved site on SA2-Scc1. Our results implicate a role for this direct antagonism in centromeric cohesion protection. Third, we report the crystal structure of human Pds5B bound to a conserved peptide motif found in both Wapl and Sororin. Further biochemical and functional studies suggest that Pds5 has both positive and negative roles in cohesion regulation and establish the molecular basis for how Wapl and the cohesin-stabilizing factor Sororin antagonistically influence cohesin dynamics on chromosomes. The structure reveals inositol hexakisphosphate (IP6) as an unexpected cofactor of Pds5. The IP6-binding segment of Pds5B engages the N-terminal region of Scc1 and inhibits the binding of Scc1 to Smc3. Our results suggest a direct role of Pds5 in cohesin release from chromosomes by stabilizing a transient, open state of cohesin during its ATPase cycle. Finally, we show that cohesin loading onto chromosomes requires the phosphorylation of MCM2-7 by Cdc7-Dbf4 kinase (DDK) during early S phase, when a mega-complex composed of MCM2-7, Scc2/4 and cohesin is formed. At active replication forks, inactivation of multiple replisome components impairs cohesin loading, weakens MCM-Scc2/4-cohesin interaction and leads to cohesion defects. By contrast, interfering Okazaki fragment processing and nucleosome assembly during DNA replication do not impact interphase cohesion, suggesting that cohesion establishment occurs before Okazaki fragment maturation and histone deposition. Our results demonstrate that DNA replication-coupled cohesin loading is required for the establishment of sister-chromatid cohesion. In conclusion, combining structural, biochemical and cellular approaches, our studies advance the molecular understanding of spatial and temporal regulation of the establishment and dissolution of sister-chromatid cohesion.