BIOL-L 211 Lecture 11 Outline of Last Lecture I. Polymerase Chain ReactionII. Agarose Gel ElectrophoresisIII. Restriction EndonucleasesOutline of Current Lecture I. Origins of ReplicationA. ProkaryotesB. EukaryotesII. Initiation of DNA Replication (E. Coli)A. Origin of ReplicationB. Initiation ProcessCurrent LectureReplication InitiationI. Origins of Replication ("ori")A. Prokaryotes1. Have only one origin of replication2. Multiple rounds of replication can occur simultaneouslya. This is important because bacteria cells divide faster than the chromosome can be replicatedb. Each bacterial daughter cell receives a chromosome that is already in the process of replicatingc. Thus, prokaryotic DNA is constantly replicating (even during segregation)3. Ori of prokaryotes has been sequenced (so it researchers can recognize the ori of prokaryotesThese 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.B. Eukaryotes1. Have hundreds to thousands of orisa. Necessary; a human chromosome is about 700x larger than an E. Coli chromosome, so it requires more oris in order to replicate efficiently2. Per cell cycle, each ori cannot initiate replication more than oncea. If occurs more than once, the result will be unnecessary duplications, potentially leading to mutations/diseases/cell death3. "Activation" of the ori = formation of replication bubble and assembly of the replisome 4. Not sequencedII. Initiation of DNA replication (E. Coli)A. Origin of Replication1. Marked by three 13-mers and five 9-mers2. 13-mer = 13 base pairs3. 9-mer = 9 base pairsB. Initiation Process1. DnaA-ATP finds 9-mer repeats and binds there (protein-DNA interaction)2. DNA separates at the 13-mer repeats3. DNA helicase (DnaB) finds the DnaA-bout region and associatesa. Requires action of DnaC (DNA helicase loader)b. DNA helicase loader analogous to sliding clamp loaderc. DNA helicase is a ring structured. Protein-protein interactions4. Helicase ring closes around ori5. Primase is drawn to the helicase, which triggers the release of DnaC and DnaA;helicase activated6. Sliding clamps (with holoenzyme complex) assemble at each primer7. DNA polymerase begins synthesizing leading strand (5' to 3')8. Sliding clamps also load DNA polymerase to the lagging strand and synthesis begins there9. Two replication forks are complete (Initiation step
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