A Systematic Study On DNA Replication Checkpoint Network In Saccharomyces Cerevisiae By E-MAP(Epistatic Miniarray Profile)

In order to ensure the accurate DNA replication,all eukaryotes have a conserved mechanism for monitoring the process of DNA replication fork.This surveillance mechanism is called the DNA replication checkpoint(also named as the S-phase checkpoint).When cells are exposed to replication stress or DNA damage,S-phase checkpoint pathway is activated to maintain genomic stability through stabilizing replication fork,slowing down the cell cycle progression,initiating DNA repair and so on.Defects in this pathway lead to genomic instability,abnormal cell cycle and tumorigenesis.Although studies during last decade have revealed the main path of the S-phase checkpoint(Mec1/ATR-Rad53/Chk2),a comprehensive network view of this pathway awaits to be obtained.In this study,we adopted a systemic method,E-MAP(epistatic miniarray profile),to explore S-phase checkpoint of Saccharomyces cerevisiae.When comparing with traditional genetic research,E-MAP has two major improvements.Firstly,via the robot operation,construction of double mutants becomes automatic and high throughput.Secondly,through imaging and measurement of respective colony size,the quantitative genetic analysis becomes feasible.We first constructed 121 single deletion mutants of query genes mainly involved in DNA replication,DNA repair and checkpoint.The library comprises 1,536 genes functioning in the cell cycle,DNA replication,and chromatin assembly.By using an antomatic ROTOR system(Singer Instruments),we successfully obtained a 121×1,536 array of double mutants.Based on the measurement of colony size,S-score calculation and stringent statistic analysis,approximately 140,000 pairs of gene interactions(GI)containing 56,891 positive and 91,251 negative interactions were identified.Our E-MAP data reach a rather high accordance rate with BioGrid data and previously reported E-MAP data,81.15%and 75.13%,respectively.43,392 genetic interactions have not been yet reported.More importantly,hydroxyurea(HU)or methyl methanesulfonate(MMS)was supplemented to examine the network rewiring under DNA replication stress or DNA damage condition.Nearly 53.40%(30,097)and 58.45%(26,966)of all GIs were affected with the treatment of HU and MMS,respectively.We next sought to identify the new components of replication checkpoint through analysis of the high throughput GI data.To this end,we focused on the dynamic response network of 9-1-1 because 9-1-1 functions as a complex in the early step of Mec1 kinase activation.By using an algorithm to calculate the genes showing significant negative interactions with all three 9-1-1 genes,we identified several candidates fomew components of S-phase checkpoint.Among them,CAC1 gene,encoding chromatin assembly complex(CAF-1)subunit was selected with the relative highest confidential value.Serial dilution experiments showed that compared with the single deletion mutants of CAC1 and any 9-1-1 subunit,their double mutants can hardly grow in the presence of MMS or HU,which can be suppressed by overexpression of deoxyribonucleic acid(RNR)reductase subunit RNR1 or RNR3.After MMS treatment,the CAC1 and 9-1-1 subunit double knockouts displayed significantly reduced Rfal foci,and Rad53 phosphorylation during S phase compared with the single mutants.Additional knockout of TEL1 gene,encoding another sensor kinase in parallel with Mecl,in cacl?ddcl?completely abolished DNA damage response.Physical interaction between CAF-1 and Mecl/Ddc2 kinase were identified through co-immunoprecipitation(Co-IP)and yeast two hybrid.Interestingly,this interaction can be enhanced by HU or MMS treatment.In vitro kinase assays showed that CAF-1 is a substrate of Mecl.All these results proved that the CAF-1 complex has an unanticipated role in the S-phase checkpoint activation.Moreover,deletion of SPT21 gene caused a defect in the activation of Rad53 as well.In summary,here we have obtained a systematic view of the S-phase checkpoint of Saccharomyces cerevisiae through drawing the high-quality "static" and "dynamic" genetic interaction maps.Through the analysis of high-throughput data,we found new important components of the S-phase checkpoint.For the first time,we report the relationship of histone chaperone CAF-1 mediated histone assembly and S-phase checkpoint activation.This study provides important clues for a more comprehensive understanding of the S-phase checkpoint and genome stability.