Studies On The Role Of Cdc45 Ubiquitination In DNA Replication

Genes support the basic structure and performance of life.Genome stability is ciucial for the faithfull inheritance of genetic information as well as the cell survival.The establishment and progression of replication forks are tightly regulated to ensure the stability of genome in the process of DNA replication.In S phase,DNA replication initiates,the replisome progression complex moves along chromatin and determines the functions of the active replication forks.In the whole process of replication,MCM2-7,GINS and Cdc45 assembled together as a complex with DNA helicase activity to unwind duplex DNA.Studies have revealed the molecular structure of the Cdc45-MCM-GINS?CMG?complex,and had indicated that Cdc45moving along chromatin is different from MCM.But how Cdc45 moves along chromatin and how the movement of Cdc45 influences the functions of CMG complex remains unknown.Ubiquitination is a kind of post-translational modification and is defined as that the ubiquitin molecules covalently bound with the target protein in the cells.Ubiquitination plays a very important role in the localization,metabolism,function,regulation and degradation of protein.Using Saccharomyces cerevisiae,we found that Cdc45 can be monoubiquitylated in this study.Bioinformatics analysis showed that Cdc45 ten potential of ubiquitylation sites.However,whether some or all of these sites can be ubiquitinated and what is the functional consequence of ubiquitylation are not clear.In this study,we found that Cdc45 can be mono-ubiquitylated when bound to the chromatin during DNA replication,and Cdc45 ubiquitylation is mediated by a ubiquitin ligase Dia2 and a deubiquitylating enzyme Doa4.We mutated potential ubiquitylation residues based on the bioinformatics analysis and found that two residues K156 and K643are essential for the ubiquitylation of Cdc45.We used FACS?fluorescene-activated cell sorting?to study the effct of Cdc45 ubiquitylation on cell cycle and found that the progression of replication fork is defective in the absense of of Cdc45 ubiquitylation or lacking Cdc45 deubiquitylation.However,simultaneous loss of Cdc45 ubiquitylation and the deubiquitylating enzyme can alleviate the defective in replication fork progression.HU is a replication inhibitor,we found that the cdc45^K156/643R and doa4?cells were significantly more sensitive to HU than the wild-type cells.Consistent with the recovering effect on replication fork progression,doa4?cdc45^K156/643R suppressed the high sensitivity to HU.We further determined the replication fork stability in the cdc45^K156/643R cells.Rad52 functions in recombinational repair and can potentially repair or restart broken or stalled replication forks.Rad52-YFP foci have been shown to form in response to replication fork collapse during the S phase.An increase in the Rad52 foci of the cdc45^K156/643R cells in S phase was observed,possibly due to damage-induced foci.Replication fork stalling at a DNA lesion generates a damage signal that activates the Rad53 kinase,which plays a vital role in survival by stabilizing stalled replication forks.In the absence of exogenous stimuli,a slower-migrating form of Rad53 was detected in the cdc45^K156/643R cells 40 min after release from G1 phase.RPA stabilizes ssDNA regions generated during DNA replication and repair,the accumulation of RPA-ssDNA complexes activates the ATR pathway.An increase in the RPA foci of the cdc45^K156/643R cells means additional ssDNA.These data indicate that the regulation of Cdc45 ubiquitylation plays an important role in maintain normal progression of replication and replication fork stability.We next found the association of MCM and GINS with chromatin was significantly decreased in the cdc45^K156/643R cells either in nomal or stressed conditions,but the level of Cdc45 was not changed.As previous reported,Cdc45 can guide the leading strand back which could slip out from the MCM central channel when the Mcm2/5 gate is open upon ATP release.We speculate that if Cdc45 in the CMG complex cannot be ubiquitylated,it would be defective in guiding leading strand back into the MCM central channel,leading to occasional dissociation of MCM from chromatin.Our findings reveal a new mechanism of regulation of the replicative integrity through Cdc45 ubiquitylation,providing critical insights into the distinct mode of Cdc45 activity at replication forks.Based on these results,we thus proposed that Cdc45 ubiquitination ensures the normal progression of the replisome along the chromatin,maintains the replication forks stability,and enables the replicative normal and integrity.We suggest that Cdc45ubiquitination provides a“replicative integrity code”.