Bio 201 1st Edition Lecture 32Outline of Last Lecture I. How Is DNA Replicated?A. Differential CentrifugationB. Density Gradient CentrifugationC. Meselson and Stahl ExperimentOutline of Current LectureI. Mechanisms of DNA ReplicationA. ChallengesCurrent LectureI. Mechanisms of DNA Replication-DNA replication is semi-conservative. The existing strands serve as a template from which to direct synthesis of the new strand. The new strand is always the reverse complement of the template strand. 3’5’ replication-Replication bubble- In bacteria, where the strands of DNA have come apart for replication. The replication bubble increases in size as replication proceeds. Arrowsreplication forks: where DNA is being actively polymerized, often called theta replication. -In bacteria, replication begins at the origin of replication (ori). There is usually one origin per chromosome.-In eukaryotes, there are multiple origins. A. Challenges1. Separating DNA Strands- DNA is held together by millions of hydrogen bonds. Pullingthese apart requires a lot of energy. Native-double helix. Denatured-Random cell. These notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.Solutions: A/T rich origins and helicase. DNA helicase separates the 2 strands, energy from ATP hydrolysis, helicases at both replication forks. 2. Initiating Synthesis of a New Strand- DNA cannot polymerize de novo from free dNTP’s: Polymerization only onto 3’ end of an existing polymer. Solutions: RNA primers. Primase catalyzes synthesis of short RNA primers at origin on both strands. The primers are complementary to the template strands. The 2 primers are oriented inopposite direction. Unlike DNA, RNA can be made de novo from free nucleotides. DNA polymerase catalyzes polymerization of DNA onto 3’ end to complete complementary strand. 3. 5’ to 3’ Problem- Polymerization only onto 3’ end of an existing polymer. Solution: Primase synthesize new RNA primers at replication forks as Helicase unwinds DNA and DNA polymerase adds nucleotides onto the 3’ end of the new primers. Results: Half of new DNA polymerization is non-continuous. Leading strand- DNA grows continuously towards replication fork. Lagging strand- DNA grows non-continuously away from replication fork. Okazaki Fragments- Bits of non-contiguous DNA in lagging strand. 4. Removal of Primers- DNA polymerase has exonuclease activity, allows it to chew up the primers from the 5’ end. DNA polymerization replaces lost RNA with DNA. Exonucleases digest RNA or DNA from the ends, one nucleotide at a time. 5. Annealing DNA ends- DNA ligase anneals the 3’ end of one DNA to the 5’ end of another. Ligases covalently attach DNA ends together. This is distinct from DNA polymerase, which use energy from hydrolysis of the triphosphorylated monomers. 6. Unwinding Supercoiled DNA- Solution: Topoisomerases have endonuclease activity that allows them to cut 1 or both strands of DNA, allowing it t unwind and thereby release tension. After unwinding, topoisomerase re-anneals the 2 strands together. This requires a lot of energy from ATP hydrolysis. Endonuclease digest RNA or DNA internally, cutting strand into
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