Structural Studies On DNA Replication Checkpoint Intermediate Protein Mrc1 And Its Assembly With DNA In Saccharomyces Cerevisiae

The living organisms are complicated.There are many kinds of living organisms on the earth,which have different shapes and characteristics.However,there are many basic common problems in the phenomena of life activities,including the basic processes of birth,growth and development,aging and death.The unique characteristics and basic life activities of each species are inseparable from the expression of genetic material.These genetic informations are basically stored in DNA-based nucleic acids.Consequently,it is very important for species continuation to preserve genetic information and transmit it to their offspring safely and unambiguously.This process depends on a complete and reliable DNA replication process.If the process of DNA replication becomes unstable or out of control,the correct transmission and expression of genetic information can not be guaranteed,and this will lead to various defects of organisms and diseases,including cancer and even death.Organisms already have a powerful and complete DNA replication system to complete the transmission of genetic material.But complex systems also have the risk of failure.In order to cope with these risks,organisms are equipped with corresponding DNA replication checkpoint mechanism and repair mechanism.Replication checkpoint maintains genomic stability of eukaryotic cells.In each cycle of genome replication,cells are inevitably challenged by endogenous or exogenous replication stress,including abnormal DNA structure,DNA damage,protein hindrance,oncogene-induced abnormal replication,inadequate supply of dNTP or histone,replication transcription conflict and so on.Replication checkpoint,also known as S-phase checkpoint,helps cells cope with these challenges and ensure the correct replication fork process before the entire genome replication.In addition to replicating genomes,DNA replication forks provide an assembly platform for many signaling proteins that function at DNA replication checkpoints.In Saccharomyces cerevisiae,Mecl is a phosphatidylinositol 3-kinase-like protein kinase(PIKK),which plays a central role in the DNA replication checkpoint pathway.It participates in the perception of stagnant DNA replication forks and is a homologous protein of ATR in mammalian cells.As a regulatory subunit of Mec1,Ddc2(mammalian ATRIP homologous protein)forms a protein complex with Mecl.Mecl-Ddc2 complexes are recruited to stagnate replication forks through single-stranded DNA coated in replication protein A(RPA).Rad1,Hus1 and Rad9 are the other three proteins required for replication checkpoints.They form trimer complexes,and their structures are thought to be similar to PCNA.The homologue of this trimer in Saccharomyces cerevisiae is Mec3-Radl-Ddc1.Another essential factor for replication checkpoints is Rad 17(Rad24 in Saccharomyces cerevisiae),which is similar to an RFC subunit and binds to RFCs2-5 to form a complex.It has been suggested that,similar to PCNA and RFCs 1-5,Rad24-RFC complexes load Mec3-Rad17-Ddcl complexes into sites where DNA damage or replication is blocked.Once loaded,the Mec3-Rad17-Ddcl complex activates Mecl.Mrcl(for mediator of replication checkpoint)is an integral part of DNA replisome and moves along chromosomes with replication forks during DNA synthesis.The deletion of MRC1 results in DNA replication defects,indicating its role in the normal progress of DNA replication.At the same time,Mrc1 also participates in the reaction of Saccharomyces cerevisiae and S.pombe cerevisiae cells to hydroxyurea.When DNA replication is blocked by hydroxyurea,Mrc1 undergoes Mec1 and Rad3(S.Pombe homologous protein of Mec1)dependent phosphorylation,which acts as an intermediary and participates in the transmission of replication stress signals from Mec1 to Rad53 effector kinase.Rad24-RFC also plays an important role in this process and may be involved in regulating the phosphorylation of Mrcl,It has been reported that phosphorylated Mrcl can increase the activation efficiency of Mecl to Rad53 by about 70 times.Although many studies have been carried out on the role of Mrcl in replication checkpoint,there is no information on the structural characteristics of Mrc1,how it interacts with DNA,how it interacts with Rad24-RFC and what factors influence the interaction between them.To solve this problem,I first attempted to purify Mrc1 from Saccharomyces cerevisiae.The genome was reconstructed by yeast homologous recombination.The target gene was tagged with TAPm,and then Mrc1 was obtained by affinity chromatography.However,due to the large number of proteins interacting with Mrc1 in vivo and the low level of background expression of Mrc1,this strategy of endogenous purification after background expression is not applicable.Luckily,we obtained the pGEX-4T-3-Mrcl plasmid as a kind gift from Lou'lab,which can express the full-length Mrcl in E.coli recombinant expression system.Also,I overexpressed the truncated Mrc1 of Saccharomyces cerevisiae by using the E.coli recombinant expression system,and obtained Mrcl(287-1096AA)in a good quality.By incubating in vitro,we obtained the complex of Mrcl(287-1096AA)and 5'flap DNA.By means of three-dimensional reconstruction of electron microscopy,we reconstructed the first negative staining electron microscopic structure of Mrcl,and realized that Mrcl is a nearly circular mediator protein,which can "grasp" DNA as part of the replisome and slide along the DNA.In addition,we recombined and expressed Mrc1(1-567AA)and incubated it with 5'flap DNA in vitro.By electron microscopy analysis,we found that the DNA affinity of truncated Mrc1,whether it is Mrcl(287-1096AA)or Mrcl(1-567AA),was significantly increased,which may be due to the exposure of the DNA binding region of truncated Mrc1.We speculate that there is an allosteric regulation between the C and N ends of Mrc1.When the interaction between the C and N ends is formed,the allosterism inhibits the affinity of DNA to the DBD on the C side.The correct and smooth replication of DNA is the key step to ensure the stability of genetic information.How the replisome binds to DNA,and how the replisome responds to the replication pressure and transmits the signal to the downstream pathway is an extremely important issue.Through electron microscopic analysis,this paper expounds the structural characteristics of Mrcl that having a hollow in the center of the ring,its binding mode with DNA that through its hollow,and its relationship with clamp loader Rad24-RFC.It has enriched people's understanding of the role of Mrcl in DNA replication and replication stress response,enriched people's understanding of replication crossover and replication checkpoint reaction.