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DNA Replication in Prokaryotes and Eukaryotes 1. Overall mechanism 2. Roles of Polymerases & other proteins 3. More mechanism: Initiation and Termination 4. Mitochondrial DNA replication DNA 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. 20.9 Does 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 ultracentrifugation. 3. Radioactivity (32P) in the DNAs of different densities is counted. Prior to 1st replication cycle, 15N (which incorporates into the bases of DNA) was added for a brief period st2nd replication cycle, cells were pulsed with 32P Prior1to (which gets incorporated into the phosphates of replicating DNA) 15N - heavy isotope of Nitrogen 32P - radioactive isotope of phosphorus DNA 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). Same Origin Random Origins Labeled DNA Labeled, sheared DNA Result: ~50% (the most possible) of the incorporated 32P was in the same DNA that was shifted by 15N Conclusion: Replication of bacterial chromosome starts at the same place every time Using 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. 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. Conclusion: eukaryotic origins also replicate bidirectionally! Fig. 20.12 in Weaver Another way to see that DNA replication is Bi-directional -Cleave replicating SV40 viral DNA with a restriction enzyme that cuts it once. Similar to Fig. 21.2 in Weaver 4 Replicon - DNA replicated from a single origin Organism # of replicons Escherichia coli (bacteria) 1 Average length of replicon 4200 kb Saccharomyces cerevisiae (yeast) Drosophila melanogaster (fruit fly) Xenopus laevis (frog) Mus musculus (mouse) 500 40 kb 3,500 40 kb 15,000 25,000 200 kb 150 kb Homo sapiens 10,000 to 100,000 Š 300 kb Eukaryotes have many replication origins. Velocity of fork movement 50,000 bp/min 3,600 bp/min 2,600 bp /min 500 bp/min 2,200 bp /min Enzymology of DNA replication: implications for mechanism 1. 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 polymerization Note: what happens to the P-P? Comparison of E.coli DNA Polymerases I and III 1 subunit 10 subunits Proofreading Activity Insertion 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 4 If 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 615 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 synthesis Lagging strand synthesis (continued) Pol III extends the RNA primers until the 3’ end of an Okazaki fragment reaches the 5’ end of a downstream Okazaki fragment. 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 doublestranded, with only one strand needing ligation, and lacking gaps. DNA Ligase Substrate Specificity Ligase will join these two G--G--A--T--C--C--T--T--G--A--T--C--C | | | | | | | | | | | | | C--C--T--A--G G--A--A--C--T--A--G--G Ligase will NOT join these two. 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--G Ligase will NOT join these two. 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--G Ligase will NOT join these two. 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--G Ligase will NOT join these two. C--C--T--A--G C--T--A--C--T--A--G--G Mechanism of Prokaryotic DNA Ligase Ligase P P HO 5' 3' NAD Ligase cleaves NAD and attaches to AMP. 1 NMN Ligase-AMP binds and attaches to 5’ end of DNA #1 via the AMP. 3' P Ligase 1 + P 3' AMP NAD The 3’OH of DNA #2 reacts with the phosphodiester shown, displacing the AMPligase. NMN +AMP 2 HO AMP + AMP & ligase separate. 2 1 3' (Euk. DNA ligase uses ATP as AMP donor) P 5' AMP Movie - Bidirectional Replication: Leading and lagging strand synthesis Other proteins needed for DNA replication: 4. DNA Helicase (dnaB gene) – hexameric protein, unwinds DNA strands, uses ATP. 5. SSB – single-strand DNA binding protein, prevents strands from re-annealing and from being degraded, stimulates DNA Pol III. 6. Gyrase – a.k.a. Topoisomerase II, keeps DNA ahead of fork from over winding (i.e., relieves torsional strain). Replisome - DNA and protein machinery at a replication fork. DNA Helicase (dnaB gene) Assay Fig. 20.21 in Weav Helicase – the movie Replication Causes DNA to Supercoil Rubber Band Model of Supercoiling DNA DNA Gyrase relaxes positive supercoils by breaking and rejoining both DNA strands.