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 ( 15 N), and one that makes it radioactive ( 32 P). 2. DNA is then isolated, sheared, and separated by CsCl density gradient ultracentrifugation. 3. Radioactivity ( 32 P) in the DNAs of different densities is counted.
Prior to 1 st replication cycle, 15 N (which incorporates into the 1 st bases of DNA) was added for a brief period Prior to 2 nd replication cycle, cells were pulsed with 32 P (which gets incorporated into the phosphates of replicating DNA) 15 N - heavy isotope of Nitrogen 32 P - 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 32 P was in the same DNA that was shifted by 15 N 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 ( 3 H-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 Average length of replicon Velocity of fork movement Escherichia coli (bacteria) 1 4200 kb 50,000 bp/min Saccharomyces cerevisiae 500 40 kb 3,600 bp/min (yeast) Drosophila melanogaster (fruit fly) 3,500 40 kb 2,600 bp /min Xenopus laevis (frog) 15,000 200 kb 500 bp/min Mus musculus (mouse) 25,000 150 kb 2,200 bp /min Homo sapiens 10,000 to 100,000 Š 300 kb Eukaryotes have many replication origins.
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 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 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 L ig ase w ill join the se t w o 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 L ig ase w ill NO T 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 L ig ase w ill NO T 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 L ig ase w ill NO T 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 L ig ase w ill NO T join these two. C--C--T--A--G C--T--A--C--T--A--G--G
Mechanism of Prokaryotic DNA Ligase 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 AMPligase. P 5' HO 3' N M N NAD 3' Ligase 1 + P AMP AMP 3' N A D AMP + NMN +AMP 1 P HO P 2 AMP & ligase separate. 3' 1 2 P 5' (Euk. DNA ligase uses ATP as AMP donor)
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.