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DNA replication Pro

DNA Replication Procaryotes

Facts about DNA replication Semi-conservative: Daughter has 1 parent and 1 new strand. Replications of both strands initate from origin sites. Specific proteins melt double helix to initiat
Helicases role in replication Use free ATP hydrolysis to unwind the duplex DNA as replication progresses
Elongation Concerns the events occuring at the replication fork, where the parent polynucleotides are copied
How do the enzymes copy DNA? In the 5' to 3' direction. The two polynucleotide strands of DNA run in opposite directions, but yet both strands grow in the same direction
What do DNA polyemerases require? A template and a primer
What is DNA error rate? 1 per 10^9 nucleotides
What needs to happen in order for DNA synthesis to begin? Both strands need to be separated. This occurs at the origin. Helicase is the key in unwinding DNA. As the strands are separated DNA is covered with single stranded binding protein.
What do single stranded binding proteins do? Prevent the reannealing of the ssDNA
DNA polymerase facts They are directional, they add nucleotides to the 3' OH of an existing primer chain extension is in the 5' to 3' direction. They are template dependent. Require a primer. Require metal ions for activity.
Primer is required...why? Primer with free 3' hydroxyl group already base-paired to the template strand. Polymerase cannot strand DNA de novo
Metal ion Mg ions bridged by carboxylate groups of two aspartate residues which hold the metal ions in the proper position and orientation. Metal ions are required for catalysis. Assist in binding of dNTP and facilitate formation of alpha phosphate and 3'OH
DNA absent, polymerase form Open form with active site on palm. Primer binds to site, dNTP forms base pair with complementary base on template. Conformational change and fingers and thumb close, polymerization is catalyzed.
Proofreading Polymerase cleaves off the mismatched nucleotides from the 3' end of the growing chain. Reaction slows down giving time for the primer terminus to be switched.
OriC 245bp. 5 repeats of a sequence that together act as a binding site for DnaA initiator protein as well as AT rich region that is more easily melted
Preprimining complex Makes single-stranded DNA accessible for other enzzymes to begin synthesis of the complementary strands. Starts with loading of helicases.
Leading strand Synthesis of one of the strands proceed in the 5' to 3' direction in a continuous manner
Okazaki fragments As the leading strand progresses, Okazaki fragments are made in a sequence that goes in the same direction as the movement of the leading strand.
Lagging strand On the lagging strand template, each fragment is laid down in a 3' to 5' direction (each fragment is made in a 5' to 3' direction). Synthesis is discontinous manner.
Describe the primer used to initiate DNA synthesis Short piece of RNa made by a primase enzyme. Short primer is extended by Pol III
Joining of the Okazaki fragments due to lagging Requires the RNA primer at the end of previous Okazaki to be removed and that eh 3'OH end of new okazaki be extended to replace the RNA primer to leave a nick that is sealed by a DNA ligase.
Nick definition Break in the phosphodiester backbone with no missing nucleotides.
DNA pol I 3'5' proofreading, 5' to 3' exonuclease activity that cleaves off nucleotides from the end of the previous okazaki. as it removes nucleotide, Pol I can insert deoxynucleotied in its place til RNA primer is removed.
DNA ligase Catalyzes the formation of a phosphodiester bond b/w 3' hydroxyl group at end of one chain and 5' phosphate group on the other. ATP required. It can only seal "nick", not a gap
Clamps Ring shaped, central cavity can accomodate DNA helix, clamp can slide freely along the DNA. Inside is - charged so they don't interact with movement of duplex.
Clamp loading enzyme All sliding clamps require clamp loading enzyme. Mulitsubunit proteins that bind to clamp and open them. then load the opened clamps onto DNA and close them. ATP required. Recognize OH of primer.
Replisome Contains Pol III enzyme, clamp, clamp loader= Pol III holoenzyme
5 subunits of clamp holder delta, delta 1, gamma and a dimer of two subunits tau.
Tau Tau monomers each bind one Pol III enzyme, thereby organizing them. Each Pol III is attached to a beta clamp. Tau binds to DnaB helicase
Pol III holoenzyme 2 Pol III molecule with their beta clamps, clamp loader, gamma complex
Topoisomerase I Binds to DNA, nicks a strand, forms a phosphodiester bond with one end of nick by attaching to tyrosine at active site. free 3' end rotates around the other strand driving by release of energy
Topoisomerase II Supercoiling. Duplex is cut, loop is passed through the cut ends and then joined.
Supercoiling Binding of 1 double helix. Each strand is positioned next to a tyrosine residue, capable of forming a covalent linkage. Complex loosly binds a 2nd DNA double helix. ATP binds and leads to comformational change
Ligation of G segment (1st DNA) LEads to release of T segment(2nd DNA) through the gate at the bottom of enzyme. Hydrolysis of ATP and release of ADP and orthophosphate allow ATP binding domains to separate, preparing to bind to another T.
DNA gyrase Is an target of several antibiotics that inhibit prokaryotic enzyme much more than eukaryotic ones. Novobiocin blocks binding of ATP to gyrase.
Nalidixic acid drug and ciprofloxacin drug Intefere with the breakage and rejoining of DNA chains. Widely used to treat urinary tract infections.
Camptothecin Antitumor agent. Inhibits human topoisomerase I by stabilizing the form of the enzyme covalently linked to DNA



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