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Bio B11 Lec 14 - Notes.doc

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Biological Sciences
Dan Riggs

Bio B11 Lec 14: DNA Replication II: enzymology, origins, organization & licensing. In the 1950’s Kornberg purified DNA polymerase (the enzyme that synthesizes new DNA strands) in E. coli. He purified an enzyme from bacterial extracts that incorporated radioactively-labeled DNA precursers into a polymer identified as DNA. Enzyme was named DNA polymerase. For the reaction to proceed it required presence of: DNA + all four deoxyribonucleosidet riphosphates dNTPs (dTTP, dATP, dCTP, dGTP) The newly synthesized radioactively labeled DNA had the same base composition as the original unlabeled DNA, suggesting the original had been used as a template. 1. Helicase: Unwinds parental Strands 2. SS DNA Binding Protein: Keeps single stranded regions unwound. 3. Primase: Initiates the strands. 4. DNA Polymerase III: Elongates chains. 5. DNA Polymerase I: Removes RNA primers and replaces them with DNA. 6. DNA Ligase: Links the short Okazaki fragments together. The role of the DNA helicase, single-stranded DNA-binding proteins, and primase at the replication fork. Helicase moves along DNA catalyzing the ATP-driven unwinding of the duplex. As strands are unwound, they’re prevented from re-forming the duplex by Single-Stranded DNA-binding Proteins (SSB’s) Then primase associated with the helicase synthesizes the RNA primers (appx 10 nucleotides long) that begin each Okazaki fragment. The RNA primers are then removed. The Origin is recognized, and unwinding & bidirectional replication begins. The replication “eye” or “bubble” forms while elongation continues and the eye expands. Each eye is made up of two replication “forks” of which each is made up of one Leading and one Lagging strand. (See Lec 13 notes for further on the Fork) Leading strand assembled continuously, lagging strand assembled as (Okazaki) fragments. Two Polymerase III molecules act to polymerize DNA 5’ to 3’ and move together in one direction. Replication of the leading and lagging strands is accomplished by two DNA polymerases working together as part of a single complex. (1) The two DNA Polymerase II molecules travel together, even though their moving towards the opposite ends of their respective templates. This is accomplished by causing the lagging strand template to form a loop. (2) the polymerase releases the lagging strand template when it encounters the previously synthesized Okazaki fragment. (3) The Polymerase that was involved in assembly of the previous Okazaki fragment has now rebound the lagging strand template farther along it's length and is synthesizing DNA onto the end of the RNA primer #3 (just constructed by primase. The lagging strand template changes size. This is the trombone model… By looping lagging strand template 180* two molecules of polymerase move in the same direction together, while both strands replicate 5'  3'. Schematic diagram of polymerase cycling on the lagging strand. The polymerase is held to the DNA by the B sliding clamp as it moves along the template strand and synthesizes the complementary strand. After Okazaki fragment completion the enzyme disengages from it's B clamp and cycles to a recently assembled clamp "waiting" at an upstream RNA primer-DNA template junction. The original B clamp is left behind for a period before it's eventually disassembled and reused. The parent strands being replicated are parallel. DNA polymerase always must go in 5' to 3' direction. The lagging strand template is looped 180* to facilitate this. There are 4 cycles of: 1:
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