DNA Replication in Prokaryotes and EukaryotesSlide 2Does DNA replication begin at the same site in every replication cycle? Electron microscope image of an E. coli chromosome being replicated. Structure (theta, θ) suggests replication started in only one place on this chromosome.Does DNA replication begin at the same site in every replication cycle?Slide 5Slide 6Slide 7Slide 8Slide 9Slide 10Slide 11Slide 12Slide 13Slide 14Slide 15Using Electron Microscopy (EM) to Demonstrate that DNA Replication is Bi-DirectionalSlide 17Slide 18Slide 19Enzymology of DNA replication: implications for mechanismSlide 21Slide 22Proofreading ActivitySlide 24Slide 25Slide 26Slide 27Slide 28Slide 29Slide 30Slide 31Slide 32Slide 33Replication Causes DNA to SupercoilRubber Band Model of Supercoiling DNADNA Replication in Prokaryotes and Eukaryotes1. Overall mechanism2. Roles of Polymerases & other proteins3. More mechanism: Initiation and Termination4. Mitochondrial DNA replicationDNA replication is semi-conservative, i.e., each daughter duplex molecule contains one new strand and one old.Does DNA replication begin at the same site in every replication cycle?Electron microscope image of an E. coli chromosome being replicated. Structure (theta, θ) suggests replication started in only one place on this chromosome.Fig. 20.9Does DNA replication begin at the same site in every replication cycle?Experiment:1. Pulse-label a synchronized cell population during successive rounds of DNA replication with two different isotopes, one that changes the density of newly synthesized DNA (15N), and one that makes it radioactive (32P).2. DNA is then isolated, sheared, and separated by CsCl density gradient ultra-centrifugation.3. Radioactivity (32P) in the DNAs of different densities is counted.1st Prior to 1st replication cycle, 15N (which incorporates into the bases of DNA) was added for a brief period Prior to 2nd replication cycle, cells were pulsed with 32P (which gets incorporated into the phosphates of replicating DNA)15N - heavy isotope of Nitrogen32P - radioactive isotope of phosphorusDNA is isolated, sheared into fragments, and separated by CsCl-density gradient centrifugation.Blow up of the last 2 rows of DNA in the previous slide (i.e., labeled DNA, and labeled, sheared DNA).Labeled DNALabeled, sheared DNASame OriginRandom OriginsConclusion:Replication of bacterial chromosome starts at the same place every timeResult: ~50% (the most possible) of the incorporated 32P was in the same DNA that was shifted by 15NUsing Electron Microscopy (EM) to Demonstrate that DNA Replication is Bi-Directional- Pulse-label with radioactive precursor (3H-thymidine)- Then do EM and autoradiography.- Has been done with prokaryotes and eukaryotes.Conclusion: eukaryotic origins also replicate bi-directionally!Drosophila cells were labeled with a pulse of highly radioactive precursor, followed by a pulse of lower radioactive precursor; then replication bubbles were viewed by EM and autoradiography.Fig. 20.12 in WeaverAnother way to see that DNA replication is Bi-directional --Cleave replicatingSV40 viral DNA with a restriction enzyme thatcuts it once. Similar to Fig. 21.2 in Weaver 4Organism # of replicons Averagelength ofrepliconVelocity offorkmovementEscherichia coli (bacteria) 1 4200 kb 50,000bp/minSaccharomyces cerevisiae(yeast)500 40 kb 3,600 bp/minDrosophila melanogaster(fruit fly)3,500 40 kb 2,600 bp/minXenopus laevis (frog) 15,000 200 kb 500 bp/minMus musculus (mouse) 25,000 150 kb 2,200 bp/minHomo sapiens 10,000 to100,000Š 300 kbReplicon - DNA replicated from a single origin Eukaryotes have many replication origins.Enzymology of DNA replication: implications for mechanism1. DNA-dependent DNA polymerases– synthesize DNA from dNTPs – require a template strand and a primer strand with a 3’-OH end– all synthesize from 5’ to 3’ (add nt to 3’ end only)Movie – DNA polymerizationNote: what happens to the P-P?Comparison of E.coli DNA Polymerases I and III1 subunit10 subunitsProofreading ActivityInsertion of the wrong nucleotide causes the DNA polymerase to stall, and then the 3’-to-5’ exonuclease activity removes the mispaired A nt. The polymerase then continues adding nts to the primer.Fig. 20.15 in Weaver 4If DNA polymerases only synthesize 5’ to 3’, how does the replication fork move directionally?• Lagging strand synthesized as small (~100-1000 bp) fragments - “Okazaki fragments” .• Okazaki fragments begin as very short 6-15 nt RNA primers synthesized by primase.2. Primase - RNA polymerase that synthesizes the RNA primers (11-12 nt that start with pppAG) for both lagging and leading strand synthesisPol III extends the RNA primers until the 3’ end of an Okazaki fragment reaches the 5’ end of a downstream Okazaki fragment.Lagging strand synthesis (continued)Then, Pol I degrades the RNA part with its 5’-3’ exonuclease activity, and replaces it with DNA. Pol I is not highly processive, so stops before going far.At this stage, Lagging strand is a series of DNA fragments (without gaps).Fragments stitched together covalently by DNA Ligase. 3. DNA Ligase - joins the 5’ phosphate of one DNA molecule to the 3’ OH of another, using energy in the form of NAD (prokaryotes) or ATP (eukaryotes). It prefers substrates that are double-stranded, with only one strand needing ligation, and lacking gaps.Ligase will join these twoG--G--A--T--C--C--T--T--G--A--T--C--C| | | | | | | | | | | | |C--C--T--A--G G--A--A--C--T--A--G--GLigase will NOT join thesetwo.G--G--A--T--C--C--T--T--G--A--T--C--C| | | | | | | | | | | |C--C--T--A--G C--A--A--C--T--A--G--GLigase will NOT join thesetwo.G--G--A--T--C--C--T--T--G--A--T--C--C| | | | | | | | | | | |C--C--T--A--A G--A--A--C--T--A--G--GLigase will NOT join thesetwo.G--G--A--T--C--C--T--T--G--A--T--C--C| | | | | | | | | | | |C--C--T--A--G G--T--A--C--T--A--G--GLigase will NOT join thesetwo.C--C--T--A--G C--T--A--C--T--A--G--GDNA Ligase Substrate Specificity21+AMP3'PAMPPAMP+HO3'P5'LigaseNAD1213'NMNHOP3'5'PLigaseNADNMN+AMPMechanism of Prokaryotic DNA Ligase Ligase cleaves NAD and attaches to AMP. Ligase-AMP binds and attaches to 5’ end of DNA #1 via the AMP.The 3’OH of DNA #2 reacts with the phosphodiester shown, displacing the AMP-ligase.AMP & ligase separate.(Euk. DNA ligase uses ATP as AMP donor)Movie - Bidirectional Replication: Leading and lagging strand synthesisReplisome - DNA and protein