Structure And Interaction Of 9-1-1 And Rad24-RFC DNA Damage Checkpoint Complexes From Saccharomyces Cerevisiae

The genome is constantly subject to stress from internal and external environments(such as ultraviolet light and ionizing radiation)causing DNA damage.Cellular DNA damage triggers cell cycle checkpoint activation,leading to a delay or arrest in cell cycle progression to prevent cell replication and inducing DNA damage repair.The clamp 9-1-1 complex and the clamp loader Rad24-RFC play an important role in cell cycle checkpoint activation.The 9-1-1 complex is a circular heterotrimeric complex composed of Rad9-Husl-Rad1,in which the C-terminus of Rad9/Ddcl contains a large number of pHospHorylation sites and protein interaction sites,including DNA damage repair key kinase ATR,TopBP1,etc.However,due to the flexibility of the Rad9/Ddcl C-terminal structure,there is currently no intact structure of the 9-1-1 complex.Obtaining the intact structure of the 9-1-1 complex under different conditions(including DNA damage conditions),especially determining the structural changes of Rad9/Ddc1,can better help us understand how the 9-1-1 complex is involved in DNA damage response.Rad24-RFC is a heteropentamer consisting of the largest subunit Rad24 and four small subunits RFC2-5.During DNA damage repair,Rad24-RFC is responsible for assisting the loading of the 9-1-1 complex into the DNA damage site,which serves to stabilize the DNA conformation and activate DNA damage checkpoints.Based on the structural similarities between the 9-1-1 complex and proliferating cell nuclear antigen(PCNA),it is currently believed that the interactions between the 9-1-1 complex and Rad24-RFC may be similar to the interactions between PCNA and RFC,but there is currently no direct evidence that the 9-1-1 complex performs a similar mechanism to Rad24-RFC.Therefore,obtaining the structural information of the intact structure of the 9-1-1 complex and the Rad24-RFC complex and its dynamic interaction will greatly help us understand the clamp loading process,but there is no structural information of the interactions between them,and even the structural information of the Rad24-RFC is rare.In this paper,the intact structure of the 9-1-1 complex in Saccharomyces cerevisiae was determined by cryo-electron microscopy(Cryo-EM).The full-length structure of the Ddc1 subunit was first analyzed and it was found that the structural flexibility of Ddc1 was finely regulated.Under normal pHysiological conditions,the 9-1-1 complex has multiple conformations and the Ddcl C-tail is highly dynamic.We obtained three relatively stable conformations,including the blocking conformation of the Ddcl C-tail in the ring region,the closed conformation of the Ddcl C-tail away from the ring region,and the open conformation of the ring opening.Methyl methanesulfonate(MMS)is a DNA damage inducer that can cause cell genome breakage.For further study,we also obtained the 9-1-1 complex under MMS induction conditions.Biochemically,the MMS-induced Ddcl subunit migrated multiple bands on the electropHoresis,and the mass spectrometry(MS)results showed that all were Ddc1.Considering that Ddc1 C-tail contains many pHospHorylation sites,it suggests that Ddcl undergoes different degrees of pHospHorylation under DNA damage conditions.At the same time,we also determined the intact structure of the MMS-induced 9-1-1 complex.Compared with the structure of the 9-1-1 complex under normal pHysiological conditions,the MMS-induced 9-1-1 complex conformation became homogeneous,Ddcl C-tail is mainly located outside the ring area.Based on biochemical and structural analysis,the structural flexibility of Ddcl C-tail is likely to be related to DNA damage response.Rad53,an effector kinase in response to DNA damage,is an important indicator of DNA checkpoint activation.Our pHospHorylation experiments revealed that Ddc1 C-tail knockout will result in complete loss or reduction of pHospHorylation of Rad53 in G1/G2 pHase.It suggests that Ddc1 C-tail plays an important role in DNA damage response.To determine the structure of Rad24-RFC,we tried endogenous expression and recombinant expression.When Rad24-RFC was endogenously purified,we found that Mecl/Ddc2(ATR/ATRIP)could be co-purified,and the Rad24-RFC/Mecl-Ddc2 complex was also found in the two-dimensional structure.In previous studies,Rad24-RFC has been considered as a clamp loader to assist the loading of the 9-1-1 complex into the DNA damage site.Once completed,Rad24-RFC will dissociate from the DNA damage.Our co-purification results may suggest that Rad24-RFC can interact directly with Mec1/Ddc2 in vivo.Due to the low concentration and purity of endogenously purified Rad24-RFC,we attempted recombinant expression to obtain Rad24-RFC samples.Under high salt and high detergent conditions,we obtained high concentration and high purity Rad24-RFC,and we also determined the complete structure of Rad24-RFC for the first time.In the negative-stained structure,the partial conformation of Rad24-RFC exhibits a "U"-shaped structure with a deep groove in the middle;the partial conformation exhibits the shape of a "claw.The clamp loader RFC complex(RFC1-5)involved in DNA replication shares four identical small subunits RFC2-5 with Rad24-RFC.Based on the crystal structure of the RFC complex,we determined the structure of the Rad24 module.At the same time,we compare the structure of RFC1 with the density of Rad24 and find that the structures are very similar.Combining the structural similarities between PCNA and the 9-1-1 complex,the similar structure of the Rad24-RFC to the RFC complex may suggest that the interaction mechanisms of the two pair of clamp-clamp loaders are similar.Based on the above structural information,we also studied the interactions between the 9-1-1 complex and Rad24-RPC,and we found that the interactions between the two are regulated by DNA damage pressure in vivo.We also successfully assembled the Rad24-RFC/9-1-1 supercomplex in vitro,which also laid the foundation for our subsequent acquisition of three-dimensional structure.