New Material DNA Replica on Cell Cycle Cancer DNA Replica on II Cellular Control End Replica on Ini0ator Proteins Complexes DNA replica on is controlled at ini a on general func ons 1 recogni on of ORI 2 may unwind duplex DNA 3 start recruitment of replisome via protein protein interac ons DNA Synthesis Occurs the S phase of Cell Cycle Overview Cell Cycle Stages of cell cycle G1 has not begun DNA synthesis stages S DNA synthesis G1 G2 M DNA synthesis has not started yet S DNA Synthesis G2 M Mitosis occurs in 24 hrs Evidence that Replication is Permitted Only Once Evidence that Replica0on is Only Permi ed Once per Cell Cycle Frog Egg M S Experiment slide 1 of 3 how to determine frequency of chromosomal DNA replica on in vivo Messelson Stahl experiment incorpora on of light heavy nucleo des into DNA biological system Xenopus egg Why use a frog egg soon aver fer liza on egg undergoes 11 divisions early embryonic cell cycles rapid w liwle or no G1 G2 called S M divisions technique microinject nuclear DNA Evidence that Replica0on is Only Permi ed Once per Cell Cycle Data Analysis slide 2 of 3 heavy DNA obtained by cell growth in medium w 15N metabolites Grow cells in medium containing 15N light DNA obtained by cell growth metabolites to produce heavy DNA i e on in medium w 14N metabolites Cesium chloride equilibrium centrifugation replica on of injected nuclear Light DNA is obtained by cell growth in 14N DNA can be monitored w a Replication of the injected nuclear DNA can density label be monitored with a density label Grow cells in medium containing 15N metabolites to produce heavy DNA i e on Cesium chloride equilibrium centrifugation Light DNA is obtained by cell growth in 14N Replication of the injected nuclear DNA can be monitored with a density label The Experiment and Results The Experiment and Results Evidence that Replica0on is Only Permi ed Once per Cell Cycle Experiment Results slide 3 of 3 Note that isotope is reversed Start with LL and add N15 as tracer RESULTS injected DNA replicates RESULTS once no HH heavy heavy DNA The injected DNA replicates ONCE detected No HH DNA was detected MULTIPLE REPLICATIONS occur if the nuclear membrane is permeabilized mul ple replica ons occur if nuclear membrane is permeabilized HYPOTHESIS an intact nuclear membrane in uences frequency of DNA replica on HYPOTHESIS An intact nuclear membrane influences frequency of DNA replication Concept Licensing of Replication Licensing Factor Model for DNA Replica0on nucleus contains ac ve LF prior to rep 1 Nucleus contains factor s that promote DNA replica on 2 Factor licensing factor consumed aver 1 round of DNA replica on 3 More factor present in cytoplasm but cannot enter nucleus 4 Licensing factor s enter nucleus when envelope breaks down NE breaks down during mitosis ac ve LF associates w nuclear material LICENSING FACTOR MODEL FOR DNA REPLICATION promote DNA replication 1 The nucleus contains a factor s that nucleus contains inac ve LF 2 The factor Licensing Factor is aver rep ac ve LF in cytoplasm 3 More of the factor is present in the can t enter cytoplasm but it cannot enter the nucleus nucleus consumed after one round of DNA replication 4 Licensing factor s enter the nucleus when the envelope breaks down Search for Licensing Factors ORC ORC is Not the Licensing Factor Licensing factor s enable ori to fire ORC was suspected to be a licensing factor but ORC is present at ori at all stages of the Cell Cycle Search for Licensing Factors ORC ORC is docking site for other replication factors ORC licensing factor LF s enable ORI to re ORC suspected to be LF but ORC is present ORI all stages of cell cycle ORC docking site for other replica on factors Expected Properties Licensing Factors Licensing Factors Proper0es proper es enable DNA synthesis S phase some e g Mcm move into nucleus end of mitosis disappear from nucleus beginning of S phase Mcm2pHMcm7p Minichromosome Maintenance proteins bind ORI ORC Mcm4p Mcm6p Mcm7p have helicase ac vity Cdc6p Cdt1p Cdc6p must bind chroma n BEFORE Cdt1p is recruited Cdt1p binds Mcm2pHMcm7p CyclinHDependent Kinases CDKs Cyclin dependent kinases Cdks APC CDKs cyclin non cataly c required subunit APC ubiqui nates mito c cyclin so cyclin can be degraded Cyclin dependent kinases Cdks APC APC Figure 17 16 Molecular Biology of the Cell Garland Science 2008 Summary of regulation Figure 17 16 Molecular Biology of the Cell Garland Science 2008 Summary of regulation CDKs APC Summary of Regula0on S phase can re o but cannot license M phase can license but cannot re o Figure 17 22 Molecular Biology of the Cell Garland Science 2008 Licensing control G1 Licensing Control G1 Phase G1 phase MCM Cdt1p and Cdc6p are loaded at Ori ORC No synthesis of DNA Mcm Cdc6p Cdt1p loaded ORI ORC Mcm2pHMcm7p binds ORI ORC Cdc6p must bind chroma n BEFORE Cdt1p is recruited Cdt1p binds Mcm2pHMcm7p no synthesis of DNA Figure 17 23 part 1 of 3 Molecular Biology of the Cell Garland Science 2008 Events at S S phase Cannot load MCM Cdt1p Cdc6p at Ori ORC Synthesis of DNA takes place MCMs leave chromatin gradually S Phase S phase Cannot load MCM Cdt1p Cdc6p at Ori ORC Synthesis of DNA takes place MCMs leave chromatin gradually cannot load Mcm Cdc6p or Cdt1p ORI ORC synthesis of DNA takes place Mcm s helicases gradually leave chroma n Cdc6p is phosphorylated degraded geminin inhibits Cdt1p Figure 17 23 part 2 of 3 Molecular Biology of the Cell Garland Science 2008 Figure 17 23 part 2 of 3 Molecular Biology of the Cell Garland Science 2008 End replication End replication S Phase ORC Ac0va0on Cdc6p Cdt1p recruit Updated View of Orc Activation at S phase Mcm helicase ac vator to promote ini a on of fork movement Cdc6 and Cdt1 recruit an activator of MCM to promote Initiation of fork movement Linear Chromosome End Replica0on The Ends of Linear Chromosomes Present a Unique Problem in DNA Replication ends of linear chromosomes present unique problem in DNA replica on each replicated strand becomes a sister chroma0d leading strand copies 5 end uses 1 primer primase has preferred sequences that may be less abundant chromosome end lagging strand is lev w a 3 overhang 3 end of lagging strand not replicated accurately by Pol Pol chromosome shortens w each replica on event possibly mutagenic Each replicated strand becomes a sister chromatid Leading strand copies 5 end easily Leading strand uses one primer Primase has preferred sequences which may be less abundant at end of chromosome Lagging strand is left with a