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Transcript of DNA Replication
* Class of enzymes that separates the two connected strands of DNA in a double helix using energy from ATP hyrdolysis. Helicase Single Strand Binding Proteins Single Strand Binding Proteins RNA Primase RNA Primase DNA Polymerase III DNA Polymerase III RNA Primer DNA Polymerase III Ligase DNA Polymerase I Ligase Ligase RNA Primer * Prevents the single strand of DNA from bonding to itself
* Increases rate of replication 100-fold. * Class of Enzymes that forms RNA primers on the lagging strand and an RNA primer on the leading strand
-Primer can only form at specific generally 2-3 Nucleotide sequences.
* Very, very slow and inefficient! * Enzyme responsible for synthesis of DNA
* Very fast and accurate!
- Replicates ~749 nucleotides per second
- Mutation rate is roughly 1.7 mutations per 10^8 nucleotides * Forms the Alpha DNA polymerase primase complex, extends from the RNA primer, forming Okazaki fragments * The enzyme that replaces the RNA with DNA * Forms a phosphodiester bond between the 5' phosphate of one Okazaki fragment with the 3' hydroxyl of another General Statements * Eukaryotes initiate this process at many different points along each DNA strand, then there are terminus site sequences of DNA, which have a Ter protein bound to them, stopping the progress of replication forks.
* Prokaryotes have circular chromosomes, so their replication stops at a terminus site, which have a Tus protein bonded to them, allowing replication forks to only pass through in one direction Base Pair Bonding * Purines bond with pyrimidines to preserve stability through distance
* A can't bond with G, and C can't bond with T. Models of DNA Replication * In 1950s, after Watson & Crick's work, 3 different models of DNA replication existed Meselson-Stahl Experiment * In nature, Escherichia Coli makes use of N^14, though N^15, a heavier isotope, also works
* Designed a system by which they could isolate and remove DNA from an E. Coli population, mix it with CsCl, place it in a centrifuge, where it will naturally settle towards bottom if its heavier, or towards the top if its lighter. Meselson-Stahl Experiment Cont'd * They grew 14 generations of E. Coli in a solution containing N^15, in the form of NH4Cl, then extracted and centrifuged DNA from that population
* The bacteria that weren't already sampled were transferred to a flask containing a solution of N^14, and allowed to multiply. The generation time of E. Coli is 20 minutes, so after every 20 minutes, DNA was extracted and centrifuged. Meselson-Stahl Experimental Results * This experiment provided the first conclusive evidence in favor of the semiconservative model.
* If it was conservative, then gen 1 would be 50% light, 50% heavy, gen 2'd be 75% light 25% heavy.
* If it was dispersive, gen 1 would be 100% intermediate, gen 2 would be 100% slightly heavier than light Polymerase Chain Reaction (PCR) * Developed by Kary Mullis in 1983 (Nobel Prize yeah!)
* Process by which a target section of DNA can be artificially copied, or "amplified", making thousands to millions of copies of a desired DNA sequence.
* Useful in forensic sciences, diagnosing of hereditary diseases, paternity testing, DNA cloning for sequencing, and detection and diagnosing of infectious diseases, among many other uses.
* Makes use of heat-stable DNA polymerases, such as TAQ polymerase, that can withstand temperature cycles that are an integral part of the process. Polymerase Chain Reaction Cont'd Polymerase Chain Reaction Cont'd * There are LOTS of different versions of this procedure, used in different circumstances.
- Can use helicase to denature instead of temperature (Helicase-dependent amplification)
- Hold the temperature at very high temps at the beginning to decrease non-specific amplification (Hot-start PCR)
- Holding at low temps at end for temporary storage (Cold-finish PCR)
- Can do multiple different target sequences (Multiplex-PCR)
- Can have one primer pair do more than one amplification (Multiplex Ligation-dependent Probe Amplification (MLPA))
- Can do multiple run-throughs of the process, reducing non-specific amplification by using one set of primers that targets the desired section and one set that targets a totally different section which are then removed at the end (Nested PCR). Physical Dynamics * As the DNA strands are being unwound and unzipped by Helicase, the force travels along the newly replicated strands of DNA, winding them up.
* However, if this process progresses too quickly, then it can result in too much DNA being rewound, before the cell had a chance to synthesize the new complementary strands. The enzymes DNA Gyrase and Topoisomerase help regulate the re-winding process, ocasionally breaking up these new strands, and giving the cell more freedom to synthesize new DNA. ? THE END! Works Cited Sadava, et al., . "The Meselson-Stahl Experiment." sumanasinc.com. Sinauer Associates, W. H. Freeman & Co., and Sumanas, Inc., n.d. Web. <http://www.sumanasinc.com/webcontent/animations/content/meselson.html>
Pearson, George. "DNA Synthesis." oregonstate.edu. Oregon University. Web. <http://oregonstate.edu/instruction/bb492/lectures/DNAI.html>.
"Polymerase Chain Reaction." dnalc.org. DNA Learning Center. Web. <http://www.dnalc.org/resources/animations/pcr.html>.
Foadey, Wilfried. "DNA Helicas: Function/Role." cs.stedwards.edu. St. Edwards University, 05 Nov 2004. Web. <http://www.cs.stedwards.edu/chem/Chemistry/CHEM43/CHEM43/Projects04/HELICASE/FUNCTION.html>.
Kahn, Jason. "DNA Ligase." Kahn Laboratory. University of Maryland, 28 Jun 2012. Web. <http://www.biochem.umd.edu/biochem/kahn/molmachines/replication/DNA Ligase.htm>.
Kahn, Jason. "Topoisomerase." Kahn Laboratory. University of Maryland, 28 Jun 2012. Web. <http://www.biochem.umd.edu/biochem/kahn/molmachines/replication/Gyrase.htm>.
DNA Replication Process. 2007. Web. <http://www.youtube.com/watch?v=teV62zrm2P0>. D-Loop Replication * Some mitochondria and chloroplasts contain a singular circular chromosome, as opposed to the more common linear chromosome, and undergo a different form of DNA replication.
* One strand of DNA is separated out from the rest of the DNA, and a complementary strand is synthesized creating a D-like structure. This process is then repeated for the other original strand of DNA Questions? Email or Facebook Us! :) Aloh Saffran