Structural Study On ATR And Its Related Complex In DNA Damage Response In Yeast

During the growth,cells encounter various extermal or internal pressures,such as radiation,chemical molecules,viruses,oxidative free radicals and so on.These stress conditions can easily cause damage to genomic DNA.DNA damage can cause many adverse eonsequences,like cell necrosis or even cancer in mammalians.To cope with the effects of these negative factors on genomic stability,eukaryotes have developed a complicated mechanism of DNA damage response to repair damaged DNA,regulate cell cycle or induce apoptosis.As a kind of important histone modification,H2B monoubiquitination(K123 in yeast and K120 in human)is widely involved in biological process such as DNA replication,gene transcription and expression,DNA damage repairing and heterochromatin maintenance.Histone modification is one of the important ways of chromatin regulation.The ubiquitination of Saccharomyces cerevisiae H2B is catalyzed by Rad6/Brel complex(ubiquitin-conjugating enzyme Rad6 and ubiquitin-ligase Brel).It remains unclear how Rad6/Brel interacts with Ubal and nuecleosome.Ubal,the ubiquitin-activating enzyme,and Rad6/Brel complex were purified from S.cerevisiae respectively.Ubal-Rad6-Bre 1 complex and nucleosome-Rad6-Bre 1 complex were assembled in vitro.Three-dimensional reconstruction revealed that Rad6/Brel stably bound on the top of Ubal.It is the first time to discover that three enzymes for ubiquitination form a stable complex.This result suggests the possibility of such a complex in vivo.It is completely different from the well-known working model of ubiquitin delivery.In the model,after ubiquitin-conjugating enzyme interacts with ubiquitin-activating enzyme and obtains ubiquitin,it interacts with ubiquitin-ligase.The ubiquitin delivery working model between Ubal,Rad6 and Brel is probably unique.In addition,Rad6/Brel can stably bind with nucleosome,demonstrating the possibility of assembling the nucleosome-Rad6-Brel complex in vitro.It lays the foundation for determining the cryo-EM structUre of nucleosome-Rad6-Brel complex in the future.In the DNA damage response(DDR)pathway,ATM(ataxia-telangiectasia mutated)and ATR(ATM and Rad3-related)kinase are the most critical regulatory proteins,which locate the most upstream of DDR signaling.ATR plays a central role in regulating single-strand DNA damage response and the stabilizing the replication fork.It is essential for cell living.High resolution cryo-EM structures of ATR/ATRIP(ATR interacting protein)from human and Mecl/Ddc2(homolog of ATR/ATRIP)from S.cerevisiae have been reported.However,the structures are in an inactive state.ATR activity is strictly regulated by a variety of factors due to the regulation of numerous cellular processes downstream.However,the interaction manner between ATR7ATRIP and cofactors is still unknown and the mechanism of ATR activation is still unclear.We reconstructed the 8.8A cryo-EM structure of Rad3-Rad26,homolog of ATR/ATRIP in Schizosaccharomyces pombe,induced by hydroxyurea(HU)in vivo.HU inhibits the dNTP pool,causing stagnation of DNA replication,which is called DNA replication stress.The overall structure of Rad3-Rad26 is butterfly shaped similar to human ATR7ATRIP and Mecl/Ddc25 but it is more flexible,especially the HEAT domain.Structure of kinase domain is more stable compared with HEAT and maintains greater conservation among different species.The in vivo activation effect of HU was confirmed by in vitro activity assay.With in vitro incubation and electron microscopy analysis,different DNA damage checkpoint complex proteins were found to activate Rad3-Rad26 in vitro in different levels and interact with Rad3-Rad26 stably as complexes.The conformation distribution of Rad3-Rad26 changes after binding to the checkpoint complex proteins.The percentage of open form is reduced and the percentage of butterfly form is increased.Especially,the complete checkpoint complex components can affect the activity and conformation distribution of Rad3-Rad26 most significantly.The synthesis and metabolism of fatty acid are closely related to the growth of cells.Fatty acid synthase(FAS)synthesizes palmitic acid and stearic acid in vivo.Recent studies have shown that ATR is involved in regulating cellular metabolism and movement,and FAS is closely related to the stability of the genome.But the relevance between ATR and FAS is still unknown.When we endogenously purified Rad3-Rad26,we found a stable co-purification of FAS by accident.By analyzing the high-resolution cryo-EM structures of co-purified FAS and the apo-state FAS,we found a significant difference on the substrate deliverer acyl carrier protein(ACP)domain of FAS.ACP stably binds to the acyltransferase(AT)domain at the entrance of substrate in the co-purified FAS.The location of the ACP domain cannot be clearly identified in the apo-state FAS.Fluorescence imaging revealed that FAS located in the cytoplasm under normal conditions or DNA damage stress.However,Rad3 existed in both nucleus and cytoplasm.Under DNA damage stress,cells with Rad3 foci in cytoplasm increased.Therefore,the interaction between Rad3 and FAS may occur in cytoplasm after Rad3 traveling out of nuclear.In this thesis,we explored the structures of ubiquitination enzymes and nucleosome.For the first time,a complex of three ubiquitination enzymes and a complex of ubiquitination enzymes with a nucleosome were obtained.It provided a new possible mechanism for ubiquitin delivery process and a basis for further exploring for the mechanism of H2B ubiquitination modification.The medium resolution cryo-EM structure of in vivo activated Rad3-Rad26?homolog of ATR/ATRIP in fission yeasty was determined.It showed a higher flexibility and differences at dimer interface.It suggests the differences between ATR in different species.The regulation of Rad3-Rad26 by the components of the checkpoint complex was studied in vitro,indicating the direction for further in vitro assembly of the checkpoint complex.We found the interaction between Rad3-Rad26 and FAS.The structure of FAS is stabilized by Rad3-Rad26.It is the first time to link fatty acid synthesis and DNA damage response together,which provides a new indication for studying ATR regulation of fatty acid synthesis and fatty acid synthase involved in DNA damage response.