BIOL-L 211 Lecture 9 Outline of Last Lecture I. Replication HypothesesII. Meselson-Stahl ExperimentIII. Methods and Minutia of DNA ReplicationOutline of Current Lecture I. DNA PolymeraseII. Sliding Clamp LoaderIII. Finishing ReplicationIV. E. Coli Holoenzyme and ReplisomeCurrent LectureReplication III. DNA PolymeraseA. Processive: Adds many dNTPs before leaving template strand1. DNA can synthesize quickly2. ~1000 dNTPs/second!!!B. Exonuclease: allows proofreading and removes improper base pairs1. Only fixes most recent errors2. Mispairing delays DNA synthesis3. Causes the primer:template junction to destabilize- moving to the exonucleaseactive siteC. Sliding clamp: donut-shaped protein complex around DNA that prevents DNA polymerase from diffusing awayThese 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.1. Without sliding clamp, DNA polymerase could only add 10-15 bases before falling off the template strand2. Does NOT interact with primaseII. Sliding Clamp Loader FunctionA. Loads the Sliding Clamps (analogous to "the claw" from Toy Story)B. ATP binds to the clamp loader, which binds and opens the sliding clampC. Sliding Clamp is placed at the primer:template junctionD. ATP hydrolysis lets clamp loader go, after which the sliding clamp closes spontaneouslyIII. Finishing ReplicationA. RNase H (endonuclease) removes most of the RNA primer1. Endonuclease: enzyme that removes nucleotides internal to DNAB. 5' Exonuclease: removes nucleotides from the end of DNAC. DNA Polymerase fills in the gap except for one little spot (which is finished by DNA ligase)D. Linear Chromosome Problems1. Lagging Strand: DNA polymerase cannot fill in the end of chromosomes without primer, which naturally leads to a gradually shrinking chromosome2. Telomeres: Ends of linear chromosomes3. Telomerase: solves the problem of the lagging strand by replicating ends of linear chromosomes (both protein and RNA)a. Binds to the lagging strand and RNA component extends as a templateb. Decreased activity with age (linked to aging)c. Werner Syndrome: premature aging caused by a mutation in WRN, a gene encoding a helicase-like protein, but also thought to helpwith DNA repair, leading to accumulation of errors and accelerated telomere shorteningIV. E. Coli Holoenzyme & ReplisomeA. Holoenzyme: complex of proteins that physically links four enzymes1. 3 DNA polymerase (one for the leading strand, two for the lagging strand)2. 1 Sliding Clamp LoaderB. Replisome: combination of proteins working together at the replication forkC. Similar process in eukaryotes, but not understood as
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