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Lecture 12

Lecture 12-DNA Replication

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Biology 1202B
Brenda Murphy

Lecture 12: DNA Replication Three Postulated Methods of DNA Replication Semi-Conservative:  The model of replication proposed by Watson and Crick  The helicase unwinds and each strand serves as a template for a new strand.  The new strands synthesized are identical to the parental strands. Conservative:  The two strands of the original molecule serve as templates for the two strands of a new DNA molecule, and then rewind into an “old” molecule.  The two copies separate from their templates to wind into the “new” molecule Dispersive:  Neither parental strand is conserved  Both chains contain old and new segments. The Semi-Conservative Model  1958 – Meselson and Stahl proved in an experiment that DNA is replicated in a semi-conservative nature.  In their experiment they had to distinguish between old parental DNA and newly replicated DNA  They did this by using “heavy” nitrogen isotope to tag parental strands 15N is heavy because it has one more neutron in its nucleus.  1.) Ecoli was grown in the heavy 15N medium for several generations, where it was incorporated into the nitrogenous bases of DNA  2.) Then they transferred the bacteria to a culture medium containing the normal light isotope 14N where the bacteria grew and divided for several generations  3.) The DNA samples were extracted into CsCl and mixed at a high rate in the centrifuge. This created a density gradient so that molecules densities separated in to bands. The densest settled closer to the bottom of the tube.  The predicated banding patterns matched the semi-conservative model method. Replication  Major steps in DNA replication: 1. The two strands of DNA unwind for replication to occur 2. Nucleotides are added only to the existing chain 3. The overall direction of new synthesis is in the 5’ to 3’ direction, which is a direction antiparallel to the template strand. 4. Nucleotides enter into a newly synthesized chain according to the A-T and G-C complimentary base-pairing rules  These are the main steps that require DNA replication. They are catalyzed by a bunch of enzymes:  DNA Helicase: Catalyzes the unwinding of the double helix, which produces a replication fork.  Replication Fork: Opens slowly and continually. A Y shape form the helical DNA and unwound template strands. o Synthesis occurs as soon as the nucleotides are exposed. o Synthesis occurs in the 5’ to 3’ orientation  Single Stranded Binding Proteins: Bind to the unwound template strands to stabilize the DNA for the replication process  Primase: Provides a short nucleotide chain, called a primer, (which is made of RNA instead of DNA) onto the DNA, because DNA polymerase can only synthesize nucleotides onto an existing strand. (It can’t start from nothing)  DNA polymerase 3: assembles new DNA to the 3’ end of RNA primers. o Can only assemble DNA in a 3’ to 5’ direction o Only one of the DNA strands runs in this direction because they are antiparallel to each other.  The Leading Strand: Runs 5’ to 3’ and is synthesized continuously  The Lagging Strand: Runs 3’ to 5’ and is synthesized discontinuously o In discontinuous replication the polymerases add short sections – called “Okazaki fragments” – of new nucleotides in the opposite direction the DNA unwinds  DNA polymerase 1: Removes the RNA primers and replaces with DNA, but the fragments are not covalently joined, there is a ‘nick’ between them  DNA Ligase: seals the nick after RNA primers are replaced with DNA so the lagging strand becomes a continuous polynucleotide sequence.  Topoisomerases: Remove over twisting and strain of DNA ahead of replication fork in a circular DNA Note – DNA replication is fast. So although process is show step by step spread out, all enzymes actually operate at the replication fork.
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