The Rtt101^Mms1-Mms22 Complex Functions In Linking Sister Chromatid Cohesion Establishment With Chromatin Replication

The accurate copying of genetic information is essential for cell growth and organism development in all eukaryotes.Faithful inheritance of the genetic information depends on chromosome replication and segregation.To ensure accurate chromosome segregation,the sister chromatids,the products of DNA replication,are kept together before they become separated prior to cell division.This physical coupling,termed sister chromatid cohesion.This process is closely related to many other important biological processes,for instance,cell cycle,DNA repair,gene transcription,chromosome formation and so on.Errors and disorders during sister chromatid cohesion may lead to incorrect chromosome segregation and result in abnormal cell cycle,genomic instability,tumorigenesis and cell death.Sister chromatid cohesion is strictly regulated.Cohesin associates with chromatin during late G1 phase in the inactive state with the help of the loading complex.The chromatin-bound cohesin becomes cohesive and physically connects two sister chromatids produced in S phase.The establishment of cohesion is largely dependent on Smc3 acetylation catalyzed by Eco1,whose activity is restricted within S phase.In anaphase,the cohesin ring is cleaved by separase and released from chromosomes.Therefore,the cohesion establishment should be coupled to DNA replication.However,the coupling molecular underpinnings have yet to be defined.We surmised that the ubiquitin ligase,Rtt101^Mms1-Mms22,may has the same function as the sister chromatid cohesion factors by analyzing the high throughput genetic interaction data in Saccharomyces cerevisiae.And the Rtt101^Mms1-Mms22 complex is known to travel along with the replication fork.Therefore,we focused on Rtt101^Mms1-Mms22,hoping to understand the mechanism of Rtt101^Mms1-Mms22 regulating sister chromatid cohesion.And then to explain the coupling mechanism between DNA replication and sister chromatid cohesion.First,by the sister chromatid cohesion assay,we noticed that lack of any Rtt101^Mms1-Mms22 subunit causes the reduced cohesion rate.Then,through yeast two hybrids and genetic analysis,we showed that Rtt101^Mms1-Mms22 interacts physically and functionally with Ecol.Mms22 directly binds to Ecolin vitro.Since Ecol is a key factor in sister chromatid cohesion,suggesting that Rtt101^Mms1-Mms22 might promote cohesion through regulating Ecol.Besides,two evolutionary conservedresidues of Ecol were mapped to be essential for interaction with Mms22.Alanine substitution of these two sites?eco1L61AG63A?impairs Smc3 acetylation and thus cohesion establishment.Taken together,the Eco1-Rtt101^Mms1-Mms22 interaction is required for efficient chromatin association of Ecol and sister chromatid cohesion.Previous reports proposed a PCNA-dependent recruitment of Ecol.To uncover the relationship between PCNA and Rtt101^Mms1-Mms22 mediated Ecol regulation,we constructed their double mutants.Growth assays showed that compared with the single deletion mutants of PCNA and any Rtt101^Mms1-Mms22 subunit,their double mutants can hardly grow at low temperature.And double mutants displayed additive loss of Smc3 acetylation.These results illustrate that Rtt101^Mms1-Mms22,together with PCNA,regulate chromatin association of Ecol and Smc3 acetylation.Meanwhile,Rtt101^Mms1-Mms22 also participates in the replication coupled nucleosome assembly.Intriguingly,our data showed that Smc3 acetylation and cohesion defects also occur in the mutants of other replication-coupled nucleosome assembly?RCNA?factors upstream or downstream of Rtt101^Mms1-Mms22.Furthermore,acetylation and cohesion defects also occur when the histone H3K121K122 ubiquitylation catalyzed by Rtt101^Mms1-Mms22 is inactivated.In summary,this study reveals a novel role of Rtt101^Mms1-Mms22 E3 in sister chromatid cohesion.These data allow us to propose a model for the coupling between nucleosome assembly and sister chromatid cohesion during DNA replication by Rtt101^Mms1-Mms22.Our study provides important clues for a more comprehensive understanding of the coupling mechanism between chromatin replication and sister chromatid cohesion.Given that these fundamental chromatin processes are highly conserved among eukaryotes,our study has important implications for medical research,diagnostic and treatment of diseases caused by chromatid cohesion defects.