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Lecture 12

Lecture 12 .docx

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Biology 1202B
Brenda Murphy

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1 DNA Replication (Chapter 12) Watson and Crick Models for Replication  Semi-Conservative (See first note)  Conservative – Each of the two strands of the original DNA serves as a template for a new DNA double helix o After the two complimentary copies separate from their templates, they wind together into an all “new” DNA double helix  Dispersive – Neither parental molecule remains in tact; both chains of each replicated double helix contain old and new segments How is it Proven that DNA Replicated in a Semi-Conservative Manner?  1958 Meselson and Stahl o Bacteria was grown for several generations in an N15 (heavy) medium  The heavy isotope, N15 has one more neutron in its nucleus than the normal N14 isotope o The heavy isotope was incorporated into the nitrogenous bases of DNA, resulting in the entire DNA being labeled with N15 o After transferring the bacteria into a medium containing light N14 isotopes, the new DNA synthesized is light o DNA mixed with Cesium chloride and centrifuged at very high speeds for 48 hours o The DNA molecules move to positions where their densities equals that of the CsCl solution and form bands o DNA of different densities is separated into bands, with the densest DNA settling closer to the bottom of the tube [N14-N14 (light), N15-N14 (hybrid), N15-N15 (heavy)] DNA Polymerase is the Primary Enzymes of DNA Replication  DNA of different densities separated into bands, with the densest DNA settling closer to the bottom of the tube  During replication, complimentary polynucleotides chains are assembled from individual deoxyribonucleotides by enzymes known as DNA polymerase  Deoxyribonucleoside triphosphates are the substrates for the polymerization reaction catalyzed by DNA polymerase  DNA polymerase catalyzes the assembly of a new DNA strand that is complimentary to the template strand  The 5’ end has an exposed phosphate group attached to the 5’ carbon of the sugar, and the 3’ end has an exposed hydroxyl group attached to the 3’ carbon of the sugar  DNA polymerase can add a nucleotide only to the 3’ end of an existing nucleotide chain o A nucleotide is added to the new strand when an incoming dNTP enters the active site carrying a base complimentary to the template strand base positioned in the active site 2  DNA polymerase extends the new DNA strand  The template strand is “read” in the 3’ to 5’ direction for this new synthesis (lagging)  The sliding DNA clamp is a protein that encircles the DNA and binds to the rear of the DNA polymerase in terms of the forward movement during replication o Function is to tether the DNA polymerase to the template strand o Makes replication more efficient as without it, the enzyme will detach from the template much quicker Molecular events of DNA replication:  Two strands of the DNA molecule unwind for replication to occur  DNA polymerase can add nucleotides only to an existing chain  The overall direction of new synthesis is on the 5’ to 3’ direction (leading), which is a direction antiparallel to that of the template strand  Nucleotides enter into a newly synthesized chain according to the A-T and G-C complementary base-pairing rules Helicase Unwind DNA for New DNA Synthesis  Origin of Replication (ori) – Small, specific sequence in the bacterial chromosome where unwinding of DNA for replication occurs  Specific proteins bind to an ori sequence, and, in turn, promote bringing of DNA helicase, which unwinds the DNA strands  The unwinding produces a Y-shaped structure called a replication fork  Single-Stranded binding proteins (SSBs) coat the exposed single-stranded DNA segments, stabilizing the DNA and keeping the two strands from pairing back together  Topoisomerase – An enzyme that cuts the DNA ahead of the replication fork, turning the DNA on one side of the break in the opposite direction of the twisting force (to relive the twisting) and rejoins the two strands together RNA Primers Provide the Starting Point for DNA Polymerase  A primer (chain of a few nucleotides) is made of RNA instead of DNA and is synthesized by the enzyme primase  Primase then leaves the template, and DNA polymerase takes over, extending the RNA primer with DNA nucleotides as it synthesizes the new DNA chain  RNA primers are removed and replaced with DNA later in replication One New DNA Strand is Synthesized Continuously; the other, Discontinuously  New DNA is synthesized continuously in the direction of the unwinding of the double helix  The template strand, runs in the opposite direction; this means that DNA polymerase has to copy it in the direction opposite to the unwinding direction o The polymerase makes this strand in short lengths that are synthesized in the direction opposite to that of DNA unwinding 3 o The short lengths produced by this discontinuous replication are then covalently linked into a single continuous polynucleotide chain  The short lengths are called Okazaki Fragments  The new DNA synthesized in the direction of DNA unwinding is called the leading strand of DNA replication (5’ – 3’) o The template strand for that strand is the leading strand template (3’ – 5’)  The strand synthesized in the opposite direction is called the lagging strand (3’ – 5’) o The template strand for that strand is the lagging strand template (5’ – 3’) Multiple Enzymes Coordinate Their Activities in DNA Replication  Primase initiates all new strands by synthesizing an RNA primer  DNA polymerase III (leading strand), the main polymerase, extends the primer by adding DNA
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