Slide 1Considering a Health Career? Utilize The Career Center.Slide 3Slide 4Protein Interactions at the Replication ForkSlide 6RNAse HE. coli Polymerase I (Pol I)Slide 9DNA LigaseDNA LigaseSlide 12Slide 13Replication is Fast!Slide 15Slide 16Slide 17Replication InitiationSlide 19Slide 20Slide 21Slide 22Slide 23Slide 24Slide 25Slide 26Slide 27Slide 28Replication InitiationReplication InitiationCell Division in E. coliE. coli DNA is MethylatedE. coli DNA is MethylatedSlide 34Slide 35Slide 36Slide 37Termination of ReplicationSlide 39Slide 40DNA Replication in EukaryotesEukaryotic Chromosomes have Multiple OriginsEukaryotes Chromosomes have Multiple OriginsEukaryotic DNA ReplicationEukaryotic DNA ReplicationSlide 46Join Pre-SOMA! *Pre-Student Osteopathic Medical Association* FREE PIZZA AND DRINKS! GUEST SPEAKER: MED SCHOOL ADMISSIONS REP FROM A.T. STILL UNIVERSITY Monday September 29th at 6pm in 257 EB We are looking for younger board members this semester!E-mail [email protected] for more info or to be put on the mailing listConsidering a Health Career?Utilize The Career Center. Health Professions Advising•Call 217-333-0820 to make a 30 minute appointment with a health professions advisor•Stop by to ask quick questions during drop-in hours on Tuesdays and Fridays 1:30-3:00pmHealth Careers Chronicle •Subscribe at http://go.illinois.edu/chronicle •Find out about on and off campus opportunities to help you become a competitive applicantHealth Professions E-mail •E-mail us at [email protected] with questionsMCB 250 Lecture 12 DNA Replication in Bacteria (Conclusion)DNA Replication in EukaryotesLeading StrandDNA PolymeraseLagging StrandDNA PolymeraseSliding Clampg-Complext-ProteinDNA HelicaseSSB BoundTo DNA5’3’3’5’3’5’3’5’3’OHPrimase5’The ReplisomeStudy Fig 9-23Protein Interactions at the Replication Fork•Helicase can contact both Primase and t•Helicase recruits Primase to the open origin•Primase lays down 5 - 10 nucleotide primer•Clamp loader recognizes primer and loads clamp•Clamp recruits a PolIII core and initiates leading strand synthesis•Helicase recruits primase repeatedly to initiate lagging strand synthesisDNA replicationAfter Passage, Pol I Removes RNA primer and Completes DNA SynthesisDNA Ligase Seals NicksOkasaki FragmentsReplisome (2xPol III) Synthesizes both Leading and Lagging Strands at Replication Fork5’3’5’3’POHNick5’3’5’3’RNAse H•Removes RNA from RNA-DNA Hybrids–“H” stands for Hybrid•Can only cleave bonds between ribonucleotides•Therefore, leaves one ribonucleotide:E. coli Polymerase I (Pol I)•Single polypeptide – 3 domains –Polymerase Activity–3’ to 5’ Exonuclease - Fix errors–5’ to 3’ Exonuclease – Remove RNA or DNA in “front” of primer•Starts at the end of the PolIII synthesized strand and removes RNA primer replacing it with DNA. Not very processive - doesn’t have to go far.•Leaves a “nick” - break in backbone in one strand.Removal of RNA primer by DNA Polymerase I (PolI)PolI is poorly processive (it’s not interacting with a clamp) so only 10-15 bases are added per binding event).DNA LigaseNick (also called a “single strand break”)5’3’ 5’3’POH5’3’ 5’3’ATPADP + PPDNA LigaseThe enzyme transfers an AMP group from ATP to the 5’ phosphate at the nick. The 3’-OH attacks the phosphate forming a phosphodiester bond and releasing AMP.The ReplisomeLeading StrandDNA PolymeraseLagging StrandDNA PolymerasesSliding Clampg-Complext-ProteinDNA HelicaseThis figure does NOT show Primase and only a little SsbStudy Fig 9-23Watch the Video on Mastering“Study Area” (next to Etext) – “Animations” – “Replication”(Check out the other videos while you are there – eg “DNA Topology” and “Topoisomerase”You can find several animations depicting DNA replication at:http://www.hhmi.org/biointeractive/dna/animations.htmlI particularly like “DNA replication (advanced detail)”AnimationsReplication is Fast!•1000 nucleotides per second•If the DNA were 1 meter in diameter:•The DNA would be 500 miles long (2 250 mile trips)•The Replisome would be the size of a FedEx Truck•It would travel at 375 miles per hour•One error every 106 miles (which will probably be repaired)Topoisomerases Relax DNA (prevent or remove positive supercoils) in Front of Replication Fork•As helicase unwinds DNA, it decreases the twist, therefore the writhe increases in front of the replisome (Lk remains constant).•If this problem is not solved DNA replication would quickly stop.•Both Type I and Type II topoisomerases can act to remove the positive supercoils and allow replication to proceed.•In E. coli, Gyrase is believed to be the most important enzyme for relaxing DNA in front of a replication fork.Fig 9-17Note that the Topoisomerase is NOT part of the replication fork machinery per seReplication machineryPositive SupercoilsTopoIINegative SupercoilsOrigin ofReplicationResolutionChromosomal InitiationTwo ReplicationForksOriCReplication Initiation•The E. coli chromosome has a single initiation site, oriC.•oriC is recognized by a specific initiator protein, DnaA. •DnaA must be bound to ATP in order to act as initiator.•Binding of DnaA-ATP begins a series of events that leads to establishment of replicaton forks.•Replication initiation is tightly controlled.Structure of oriCThe oriC region is about 250 bps long. The 9-mer sites bind DNA A-ATP causing the 13-mer region to melt.Like Fig 9-26Figure 9-27aDnaA-ATP Binds to its Recognition Sites in OriCFigure 9-27bThe DnaA-ATP Nucleoprotein Complex Causes Unwinding of the DNAFigure 9-27dDnaC Loads DnaB (Helicase) onto the Single Stranded DNA – One Helicase on Each StrandFigure 9-27eEach Helicase Recruits a Primase to Initiate DNA SynthesisFigure 9-27fA Clamp Loader Recognizes the 3’ End and Loads a Clamp on Each StrandFigure 9-27gThe Two Replisomes are Now Formed and Proceed Around the Chromosome5’3’5’3’Clicker QuestionWhich direction is this replication fork traveling?A.B.5’3’5’3’Clicker QuestionWhich is the leading strand template?AB5’3’5’3’Clicker QuestionWhich is the leading strand template of the right fork?ABReplication Initiation5’3’ 5’3’OkazakiFragmentsOrigin“Red” Replisome“Black” ReplisomeOriginLeadingLagging LeadingLagging•The Primase in Each Replisome can Only Prime the Lagging Strand•The First Lagging